Bicarbonate as a potentiator for antimicrobial agents

ABSTRACT

Provided herein are methods and compositions for modulating a microorganism&#39;s response to an antimicrobial agent. In one embodiment, the method comprises contacting the microorganism with an antimicrobial agent and bicarbonate. In one embodiment, provided herein are methods for treating a microbial infection comprising administering to a subject in need an effective amount of (i) bicarbonate and (ii) an antimicrobial agent. Also provided herein are methods of screening for antimicrobial compounds.

This application is a division of U.S. patent application Ser. No.15/887,469, filed Feb. 2, 2018, now U.S. Pat. No. 10,940,163, whichclaims the benefit of U.S. Provisional Patent Application No.62/524,866, filed Jun. 26, 2017, U.S. Provisional Patent Application No.62/483,032, filed Apr. 7, 2017 and U.S. Provisional Patent ApplicationNo. 62/453,701, filed Feb. 2, 2017, each of which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present application relates to the activity of bicarbonate as amodulator of antimicrobial agents, and in particular the use of sodiumbicarbonate as a modulator of antibiotics.

BACKGROUND OF THE INVENTION

There has been a steady increase in the use of antimicrobial agents,such as antibiotics and anti-fungal agents. Over the past 20 years,there has been an explosion in the prevalence of antibiotic resistantbacterial infections, both in the hospital and in the general community.Notably, the ESKAPE pathogens (Enterococcus faecium, Staphylococcusaureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonasaeruginosa, and Enterobacter species) are responsible for a substantialpercentage of nosocomial infections and present serious therapeuticchallenges for physicians. These multi-drug resistant infectionsincrease morbidity and mortality and undoubtedly, require broad-spectrumantimicrobial coverage, or the increased usage of antibiotics.

Needed are ways than can further modulate the response of microorganismsto various antimicrobial agents, in order to achieve control of activityof antimicrobial agents, useful in various contexts.

For example, needed are methods to potentiate the response of amicroorganism to an antimicrobial agent, allowing for either a greaterresponse at a given concentration of the antimicrobial agent, or anincreased response at a lower concentration of the antimicrobial agent.This could, for example, establish a way by which to preserve theefficacy of existing antibiotics that may work better in a host thanoriginally thought or a means to afford novel and alternate clinicalindications.

SUMMARY OF THE INVENTION

The invention provides methods for inhibiting the growth of a virus, abacterium, a fungus or a parasite, comprising contacting the virus, thebacterium, the fungus or the parasite with an effective amount of (i)bicarbonate and (ii) an antimicrobial agent;

wherein the bacterium is:

(a) a spiral-shaped bacterium, a filamentous bacterium, a pleomorphicbacterium or a rectangular bacterium;

(b) an obligate aerobe or an obligate anaerobe;

(c) a Gram positive rod-shaped bacterium;

(d) a Gram negative sphere-shaped bacterium;

(e) a species of Acinetobacter, Actinomyces, Aerococcus, Agrobacterium,Anaplasma, Azorhizobium, Azotobacter, Bacillus, Bacteroides, Bartonella,Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium,Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium,Coxiella, Ehrlichia, Enterobacter, Enterococcus, Francisella,Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella,Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium,Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma,Neisseria, Pasteurella, Pediococcus, Peptostreptococcus, Porphyromonas,Prevotella, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia,Salmonella, Serratia, Shigella, Sarcina, Spirillum, Spirochaetes,Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus,Treponema, Vibrio, Viridans, Wolbachia or Yersinia; or

(f) Staphylococcus epidermidis.

The invention also provides methods for treating or preventing amicrobial infection, comprising administering to a subject in needthereof an effective amount of (i) bicarbonate and (ii) an antimicrobialagent;

wherein the microbial infection is an infection by a virus, a bacterium,a fungus or a parasite; and

wherein the bacterium is:

(a) a spiral-shaped bacterium, a filamentous bacterium, a pleomorphicbacterium or a rectangular bacterium;

(b) an obligate aerobe or an obligate anaerobe;

(c) a Gram positive rod-shaped bacterium;

(d) a Gram negative sphere-shaped bacterium;

(e) a species of Acinetobacter, Actinomyces, Aerococcus, Agrobacterium,Anaplasma, Azorhizobium, Azotobacter, Bacillus, Bacteroides, Bartonella,Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium,Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium,Coxiella, Ehrlichia, Enterobacter, Enterococcus, Francisella,Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella,Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium,Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma,Neisseria, Pasteurella, Pediococcus, Peptostreptococcus, Porphyromonas,Prevotella, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia,Salmonella, Serratia, Shigella, Sarcina, Spirillum, Spirochaetes,Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus,Treponema, Vibrio, Viridans, Wolbachia or Yersinia; or

(f) Staphylococcus epidermidis.

The invention further provides methods for inhibiting the growth of avirus, a bacterium, a fungus or a parasite, comprising contacting thevirus, the bacterium, the fungus or the parasite with an effectiveamount of (i) bicarbonate and (ii) an antimicrobial agent;

wherein the antimicrobial agent is an antiviral agent, an anti-fungalagent, an anti-parasitic agent, an antibiotic agent, or an innateimmunity factor;

wherein the antibiotic agent is

(a) a macrolide, a tetracycline, a cephalosporin, a quinolone, arifampin or a fluoroquinolone; or

(b) Amoxicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin,Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin,Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin,Ticarcillin, or a pharmaceutically acceptable salt thereof; and

wherein the innate immunity factor is an antimicrobial enzyme or anantimicrobial secretion.

The invention further provides methods for treating or preventing amicrobial infection, comprising administering to a subject in needthereof an effective amount of (i) bicarbonate and (ii) an antimicrobialagent;

wherein the microbial infection is an infection by a virus, a bacterium,a fungus or a parasite;

wherein the antimicrobial agent is an antiviral agent, an anti-fungalagent, an anti-parasitic agent, an antibiotic agent, or an innateimmunity factor;

wherein the antibiotic agent is

(a) a macrolide, a tetracycline, a cephalosporin, a quinolone, arifampin or a fluoroquinolone; or

(b) Amoxicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin,Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin,Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin,Ticarcillin, or a pharmaceutically acceptable salt thereof; and

wherein the innate immunity factor is an antimicrobial enzyme or anantimicrobial secretion.

The invention further provides methods for modulating a microorganism'sresponse to an antimicrobial agent, comprising contacting amicroorganism with an antimicrobial agent in the presence ofbicarbonate, wherein the antimicrobial agent is an antibiotic, andwherein the antibiotic is a macrolide, tetracycline, peptide,glycopeptide, penicillin, cephalosporin, quinolone, fluoroquinolone,rifampin, an innate immunity factor or an antifungal agent.

The invention further provides methods for modulating a microorganism'sresponse to an antimicrobial agent, comprising contacting amicroorganism with an antimicrobial agent in the presence ofbicarbonate, wherein the antimicrobial agent is not an aminoglycoside,kanamycin, gentamicin, LL-37, or pharmaceutically acceptable saltsthereof.

The invention further provides methods for modulating a microorganism'sresponse to an antimicrobial agent, comprising contacting amicroorganism with the antimicrobial agent in the presence ofbicarbonate, wherein the antimicrobial agent is pentamidine,indolicidin, bactenesin, alpha-defensin, bile salts, lysozyme,protegrin, hyaluronic acid, chloramphenicol, dirithromycin,erythromycin, doxycycline, tetracycline, linezolid, bacitracin,fosfomycin, fosmidomycin, ampicillin, amoxicillin, cloxacillin,piperacillin, oxacillin, ceftriaxone, cefoperazone, vancomycin,polymyxin B, ciprofloxacin, besifloxacin, enoxacin, nalidixic acid,norfloxacin, levofloxacin, moxifloxacin, pefloxin, novobiocin,rifampicin, trimethoprim or sulfamethoxazole, or a pharmaceuticallyacceptable salts thereof.

The invention further provides methods for modulating a microorganism'sresponse to an antimicrobial agent comprising contacting a microorganismwith an antimicrobial agent in the presence of bicarbonate, wherein theantimicrobial agent is pentamidine, indolicidin, bactenesin,alpha-defensin, bile salts, lysozyme, protegrin, hyaluronic acid,apramycin, neomycin, paromycin, spectinomycin, chloramphenicol,dirithromycin, erythromycin, doxycycline, tetracycline, linezolid,bacitracin, fosfomycin, fosmidomycin, ampicillin, amoxicillin,cloxacillin, piperacillin, oxacillin, ceftriaxone, cefoperazone,vancomycin, polymyxin B, ciprofloxacin, besifloxacin, enoxacin,nalidixic acid, norfloxacin, levofloxacin, moxifloxacin, pefloxin,novobiocin or rifampicin, or a pharmaceutically acceptable salt thereof.

The invention further provides compositions comprising: an effectiveamount of (i) bicarbonate and (ii) an antimicrobial agent.

The invention further provides compositions comprising:

an effective amount of (i) bicarbonate and (ii) an antimicrobial agent;

wherein the antimicrobial agent is an antiviral agent, an antibioticagent, an anti-fungal agent, an anti-parasitic agent or an innateimmunity factor; and

wherein the antibiotic agent is

(a) a macrolide, a tetracycline, a cephalosporin, a quinolone or afluoroquinolone; or

(b) Amikacin, Neomycin, Tobramycin, Paromomycin, Streptomycin,Spectinomycin, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem,Cefadroxil, Cefazolin, Cefalothin, Cefalexin, Cefaclor, Cefamandole,Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren,Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten,Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole,Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin,Lipopeptide, Daptomycin, Azithromycin, Clarithromycin, Dirithromycin,Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin,Aztreonam, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin,Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin,Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, PenicillinV, Piperacillin, Penicillin G, Temocillin, Ticarcillin, Bacitracin,Colistin, Polymyxin B, Besifloxacin, Enoxacin, Gatifloxacin,Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid,Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin,Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silversulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole,Sulfanilimide, Sulfasalazine, Sulfisoxazole, Sulfonamidochrysoidine,Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline,Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole,Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol,Tigecycline, Tinidazole, Trimethoprim, Clofazimine, Dapsone,Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid,Pyrazinamide, Rifampicin, Rifabutin, Rifapentine or Streptomycin, or apharmaceutically acceptable salt thereof.

The invention further provides methods of potentiating the activity ofan antimicrobial agent, comprising administering to a subject in needthereof an effective amount of (i) bicarbonate and (ii) an antimicrobialagent, wherein the antimicrobial agent is an antimicrobial agent whoseconcentration in a cell of the microorganism is increased by one or bothof (1) decrease in pH gradient across the microorganism's cytoplasmicmembrane; and (2) increase in the microorganism's cytoplasmic membranepotential.

The invention further provides compositions comprising sodiumbicarbonate and an antimicrobial agent wherein the antimicrobial agent'sconcentration in a cell of the microorganism is increased by one or bothof (1) decrease in pH gradient across the microorganism's cytoplasmicmembrane; and (2) increase in the microorganism's cytoplasmic membranepotential.

The invention further provides methods of treating a microbialinfection, or a disease, disorder or condition arising from a microbialinfection, comprising administering, to a subject in need thereof, aneffective amount of (i) bicarbonate and (ii) an antimicrobial peptide,an antimicrobial enzyme, or an antimicrobial secretion.

The invention further provides compositions comprising sodiumbicarbonate and an antimicrobial peptide, an antimicrobial enzyme, or anantimicrobial secretion.

The invention further provides methods for treating a microbialinfection, or a disease, disorder or condition arising from a microbialinfection, comprising administering, to a subject in need thereof, aneffective amount of (i) bicarbonate and (ii) an antimicrobial agent,wherein the antimicrobial agent targets the membrane potential of acell.

The invention further provides methods for screening for antimicrobialcompounds comprising: (1) contacting a microorganism with a testcompound in the presence of bicarbonate; and (2) observing growth of themicroorganism, wherein a decrease in the growth of the microorganism inthe presence of the test compound compared to in the absence of the testcompound indicates that the test compound is an antimicrobial compound.

The invention further provides methods for screening for anantimicrobial compound that can be modulated by bicarbonate comprising:(1) contacting a microorganism with a test compound (i) in the presenceof bicarbonate or (ii) not in the presence of bicarbonate; and (2)observing growth of the microorganism, wherein a greater change in thegrowth of the microorganism with the test compound in the presence ofbicarbonate compared to the growth of the microorganism with the testcompound not in the presence of bicarbonate indicates that the testcompound is an antimicrobial compound that can be modulated bybicarbonate.

Other features and advantages of embodiments provided herein will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating embodiments of the application, are given byway of illustration only and the scope of the claims should not belimited by these embodiments, but should be given the broadestinterpretation consistent with the description as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are shown in more depth by the followingdescriptions of their respective drawings listed below.

FIG. 1A shows graphically that pentamidine possesses antibacterialactivity alone and in the presence of novobiocin.

FIG. 1B shows graphically that pentamidine alone and in the presence ofnovobiocin causes clearance of bacteria from the spleen.

FIG. 2 shows checkerboard microdilution assays and demonstrates, in anillustrative embodiment, the effect of added sodium bicarbonate onpentamidine activity against E. coli, where dark regions representhigher growth. A synergistic interaction is observed.

FIG. 3A and FIG. 3B show the dissipation of the bacterial membranepotential by pentamidine without (A) or with (B) the addition of sodiumbicarbonate, in an illustrative embodiment of the invention.

FIG. 4A and FIG. 4B graphically demonstrates the bacterial loadfollowing infection in tape-stripped mice, with and without pentamidinetopical treatment, in an illustrative embodiment of the invention.

FIG. 5 shows the effect of a topical pentamidine-sodium bicarbonateformulation on bacterial load following infection in tape-stripped mice,in an illustrative embodiment of the invention.

FIG. 6A shows the potentiation or suppression of antibiotics against E.coli in the presence of 25 mM sodium bicarbonate, an illustrativeembodiment of the invention. FIG. 6B shows similarly that bicarbonateaffects the activity of various classes of antibiotics in E. coli and S.aureus. FIG. 6C shows a microdilution checkerboard analyses forantibiotics when used with sodium bicarbonate against E. coli.

FIG. 7 shows the suppression of tetracycline entry in the presence ofvarying concentrations of sodium bicarbonate, in an illustrativeembodiment of the invention.

FIG. 8A shows the potentiation of the activity of dirithromycin in thepresence of 25 mM sodium bicarbonate and FIG. 8B shows the disappearanceof potentiation in a mutant of E. coli lacking the efflux pump tollC, anillustrative embodiment of the invention. FIG. 8C, Panels a-c show thecombination of the macrolide, dirithromycin, and sodium bicarbonateagainst multi-drug resistant clinical isolates of (a) Acinetobacterbaumannii (b) Klebsiella pneumoniae and (c) Pseudomonas aeruginosa.

FIG. 9 shows a graph showing that the MIC of pentamidine used againstSaccharomyces cerevisiae is reduced from 256 μg/mL to 4 μg/mL in thepresence of 25 mM sodium bicarbonate and to 2 μg/mL in the presence of50 mM sodium bicarbonate in an illustrative embodiment of the invention.

FIG. 10 shows that physiological concentrations of bicarbonate enhancethe antibacterial activity of various chemical factors involved ininnate immunity. Shown are potency analyses of various componentsagainst E. coli in MHB (line with Xs) and MHB supplemented with 25 mMsodium bicarbonate (line with circles) for a, LL-37; b, indolicidin; c,bactenesin; d, alpha-defensin; e, bile salts; f, lysozyme; g, protegrin;and h, hyaluronic acid.

FIG. 11 , Panels a-c show that bicarbonate dissipates the pH gradientacross the cytoplasmic membrane affecting the activity of antibiotics.

FIG. 12 shows uptake of 3,3′-Dipropylthiacarbocyanine iodide, amembrane-potential sensitive dye, in S. aureus cells in the absence(untreated, white circles) or presence of 25 mM sodium bicarbonate(treated, black circles).

FIG. 13 shows that a combination of nigericin, a protonophore, withfosfomycin (Panel a) or novobiocin (Panel b) leads to antagonisticinteractions against S. aureus. Shown are microdilution checkerboardanalyses, where the extent of inhibition is shown as a heat plot, suchthat the darkest color represents full bacterial growth.

FIG. 14 shows that sodium bicarbonate inhibits cellular respiration inE. coli.

FIG. 15 shows the growth curve of E. coli grown in the presence ofvarying concentrations of sodium bicarbonate.

FIG. 16 shows the effect of 25 mM sodium bicarbonate on intracellularATP levels, measured by a luciferin-luciferase bioluminescence assay.

FIG. 17 , Panels a-b show the chemical-genomic interactions with 25 mMbicarbonate in E. coli K12.

FIG. 18 , Panels a-c show microdilution checkerboard analyses for sodiumbicarbonate and molecules shown to dissipate ΔΨ.

FIG. 19 , Panels a-b show the effect of pH-adjusting media on thecombination of dirithromycin with trisodium phosphate.

FIG. 20 shows changes in intracellular pH upon treatment withbicarbonate.

FIG. 21 , Panels a-b show the growth inhibition by dirithromycin andsodium bicarbonate. Bacterial strains were wild type E. coli in Panel a;and ΔychM in Panel b.

FIG. 22 shows microdilution checkerboard analyses for the effect ofvaried sodium bicarbonate concentrations on the activity of variousantibiotics against clinical isolates of various bacteria. Dark regionsrepresent higher growth of the microorganism. “MRSA” refers tomethicillin-resistant Staphylococcus aureus. “MSSA” refers tomethicillin-sensitive Staphylococcus aureus.

FIG. 23 shows microdilution checkerboard analyses for the effect ofvaried sodium bicarbonate concentrations on the activity of pentamidineagainst various bacteria. Dark regions represent higher growth of themicroorganism.

FIG. 24 shows microdilution checkerboard analyses for the effect ofvaried sodium bicarbonate concentrations on the activity of variousmacrolides against methicillin-resistant Staphylococcus aureus (MRSA).Dark regions represent higher growth of the microorganism.

FIG. 25 shows microdilution checkerboard analyses for the effect ofvaried sodium bicarbonate concentrations on the activity of variousmacrolides against various clinical isolates of P. aeruginosa. Darkregions represent higher growth of the microorganism.

DETAILED DESCRIPTION

Provided herein are compositions and methods for modulating amicroorganism's response to an antimicrobial agent. The methods comprisecontacting a microorganism with an antimicrobial agent in the presenceof bicarbonate, whereby the bicarbonate modulates the microorganism'sresponse to the antimicrobial agent.

Also provided herein are compositions and methods for treating amicrobial infection, or a disease, disorder or condition arising from amicrobial infection. The treatment methods comprise administering to asubject in need thereof, an effective amount of (i) bicarbonate and (ii)an antimicrobial agent.

Also provided herein are methods of screening for antimicrobial agentscomprising (1) contacting a microorganism with a test compound in thepresence of bicarbonate; and (2) observing growth of the microorganism,wherein a decrease in the growth of the microorganism in the presence ofthe test compound compared to in the absence of the test compoundindicates that the test compound is an antimicrobial agent.

Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the present invention herein described for which they aresuitable as would be understood by a person skilled in the art.

The term “substantially”, “about” and “approximately”, when immediatelypreceding a number or numeral, means that the number or numeral rangesplus or minus 10%, and in some embodiments plus or minus 5%.

In embodiments comprising an “additional” or “second” component, such asan additional or second antibiotic, the second component as used hereinis chemically different from the other components or first component. A“third” component is different from the other, first, and secondcomponents, and further enumerated or “additional” components aresimilarly different.

The term “microbial infection” as used herein refers to an invasion ofcells or bodily tissues by a foreign, undesirable microorganism.

The term “subject” as used herein includes all members of the animalkingdom including mammals, and in some embodiments, to humans. Thus, themethods provided herein are applicable to both human therapy andveterinary applications. In some embodiments, the subject is a mammal.In another embodiment, the subject is human.

The term “pharmaceutically acceptable” means compatible with thetreatment of subjects, for example humans.

The term “pharmaceutical composition” as used herein refers to acomposition that is suitable for pharmaceutical use.

The term “pharmaceutically acceptable carrier” means a non-toxicsolvent, dispersant, excipient, adjuvant or other material which ismixed with the active ingredient in order to permit the formation of apharmaceutical composition, i.e., a dosage form capable ofadministration to a subject. One non-limiting example of such a carrieris a pharmaceutically acceptable oil typically used for parenteraladministration.

The term “pharmaceutically acceptable salt” means either an acidaddition salt or a base addition salt which is suitable for, orcompatible with the treatment of subjects.

An acid addition salt suitable for, or compatible with, the treatment ofsubjects is any non-toxic organic or inorganic acid addition salt of anybasic compound. Basic compounds that form an acid addition salt include,for example, compounds comprising an amine group. Illustrative inorganicacids which form suitable salts include hydrochloric, hydrobromic,sulfuric, nitric and phosphoric acids, as well as acidic metal saltssuch as sodium monohydrogen orthophosphate and potassium hydrogensulfate. Illustrative organic acids which form suitable salts includemono-, di- and tricarboxylic acids. Illustrative of such organic acidsare, for example, acetic, trifluoroacetic, propionic, glycolic, lactic,pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric,ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic,cinnamic, mandelic, salicylic, 2-phenoxybenzoic, isethionic,p-toluenesulfonic acid and other sulfonic acids such as methanesulfonicacid, ethanesulfonic acid and 2-hydroxyethanesulfonic acid. Either themono- or di-acid salts can be formed, and such salts can exist in eithera hydrated, solvated or substantially anhydrous form. In general, acidaddition salts are more soluble in water and various hydrophilic organicsolvents, and generally demonstrate higher melting points in comparisonto their free base forms. Other non-pharmaceutically acceptable saltsinclude but are not limited to oxalates.

A base addition salt suitable for, or compatible with, the treatment ofsubjects is any non-toxic organic or inorganic base addition salt of anyacidic compound. Acidic compounds that form a basic addition saltinclude, for example, compounds comprising a carboxylic acid group.Illustrative inorganic bases which form suitable salts include lithium,sodium, potassium, calcium, magnesium or barium hydroxide as well asammonia. Illustrative organic bases which form suitable salts includealiphatic, alicyclic or aromatic organic amines such as isopropylamine,methylamine, trimethylamine, picoline, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, EGFRaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins, and the like.Illustrative organic bases are isopropylamine, diethylamine,ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.[See, for example, S. M. Berge, et al, “Pharmaceutical Salts,” J. Pharm.Sci. 1977, 66, 1-19]. The selection of the appropriate salt may beuseful so that an ester functionality, if any, elsewhere in a compoundis not hydrolyzed. The selection criteria for the appropriate salt willbe known to one skilled in the art.

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid orbase in a suitable solvent and the formed salt is isolated byfiltration, extraction or any other suitable method.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” means an amount effective, at dosages and/or forperiods of time necessary to achieve a desired result.

The term “bicarbonate” as used herein refers to a compound of theformula XHCO₃, wherein X is a suitable cation. In some embodiments,“bicarbonate” refers to HCO₃ ⁻ together with a cation. In someembodiments, a cation is an alkali metal cation. For example, a cationmay be sodium, lithium or potassium. In some embodiments, a cation is analkaline earth metal cation. For example, a cation may be magnesium orcalcium. In other embodiments, bicarbonate is ammonium bicarbonate orzinc bicarbonate.

The expression “cytoplasmic membrane potential” as used herein refers todifference in electric potential between the interior and the exteriorof a biological cell.

The expression “proton motive force” and “PMF” as used interchangeablyherein refers to the measure of the potential energy stored as acombination of proton and voltage (electrical potential) gradientsacross a membrane. The electrical gradient is a consequence of thecharge separation across the membrane (when the protons H⁺ move withouta counterion, such as chloride Cl⁻).

The expression “psi component of the proton motive force (PMF)” or Δψ,as used herein refers to the transmembrane electrical potential, thedifference in electrical potential across a membrane.

The term “aminoglycoside” as used herein refers to a class of antibioticagents that inhibit bacterial protein synthesis and contain, as aportion of the molecule, an amino-modified glycoside (sugar).Aminoglycosides include, but are not limited to, apramycin, gentamicin,kanamycin, neomycin, paromycin, and spectinomycin.

As used herein, the “minimal inhibitory concentration” or “MIC” is thelowest detectable concentration that completely inhibits microbialgrowth under standard growth conditions.

The term “microorganism”, as used herein, refers to an organism that ismicroscopic. Microorganisms include single-celled and multicellularorganisms. Microorganisms include bacteria, archaea, protozoa, algae,fungi, viruses, and multicellular animal parasites (helminths).

The term “potentiating”, as used herein, refers to increasing the effectof an antimicrobial agent on a microorganism.

The term “modulating”, as used herein, refers to changing (potentiatingor lessening) the effect of an antimicrobial agent on a microorganism.

Antimicrobial Agents

The methods provided herein comprise contacting a microorganism with anantimicrobial agent in the presence of bicarbonate. Also provided hereinare compositions comprising (a) an effective amount of (i) bicarbonateand (ii) an antimicrobial agent; and, optionally, (b) a pharmaceuticallyacceptable carrier, diluent or excipient. In some aspects of the methodsand compositions described herein, an antimicrobial agent is anantiviral agent, an antibiotic agent, an anti-fungal agent, ananti-parasitic agent or an innate immunity factor. In some embodiments,the methods comprise contacting a microorganism with (i) bicarbonate and(ii) a plurality of different antimicrobial agents.

In some embodiments, the antimicrobial agent causes an increase in thepH gradient across the microorganism's cytoplasmic membrane. In someembodiments, the antimicrobial agent causes a decrease in the pHgradient across the microorganism's cytoplasmic membrane. In someembodiments, the antimicrobial agent causes an increase in themicroorganism's cytoplasmic membrane potential. In some embodiments, theantimicrobial agent is a cationic antimicrobial agent, an antimicrobialagent that is an energy dependent efflux substrate, an antimicrobialagent for which entry depends on membrane potential, or an antimicrobialagent that disrupts membrane potential as a primary mechanism of action.

Antibiotics

In some embodiments, an antimicrobial agent is an antibiotic. The terms“antibiotic”, “antibiotic agent” and “antibacterial agent” may be usedinterchangeably.

In some embodiments, the antibiotic is a macrolide, an aminoglycoside, atetracycline, a peptide, a glycopeptide, a penicillin, a cephalosporin,a quinolone, a fluoroquinolone or a rifampin. An antibiotic may also bea pharmaceutically acceptable salt of any molecule described above, or acombination of these molecules.

In some embodiments, the antibiotic is an aminoglycoside. In someembodiments, the antibiotic is apramycin, gentamicin, kanamycin,neomycin, paromycin, spectinomycin, a combination thereof, or apharmaceutically acceptable salt thereof. In some embodiments, theantibiotic is not kanamycin or a pharmaceutically acceptable saltthereof. In some embodiments, the antibiotic is not gentamicin or apharmaceutically acceptable salt thereof. In some embodiments, theantibiotic is not pentamidine or a pharmaceutically acceptable saltthereof.

In some embodiments, the antibiotic is not an aminoglycoside or apharmaceutically acceptable salt thereof.

In some embodiments the antibiotic is a macrolide. In some embodiments,the antibiotic is dirithromycin, erythromycin, a combination thereof, ora pharmaceutically acceptable salt thereof.

In some embodiment, the antibiotic is not a macrolide or apharmaceutically acceptable salt thereof.

In some embodiments the antibiotic is a tetracycline or apharmaceutically acceptable salt thereof. In some embodiments theantibiotic is doxycycline, tetracycline, a combination thereof, or apharmaceutically acceptable salt thereof.

In some embodiments the antibiotic is not a tetracycline or apharmaceutically acceptable salt thereof.

In some embodiments the antibiotic is a penicillin or a pharmaceuticallyacceptable salt thereof. In some embodiments, the antibiotic isampicillin, amoxicillin, cloxacillin, piperacillin, oxacillin, acombination thereof, or a pharmaceutically acceptable salt thereof.

In some embodiments the antibiotic is not a penicillin or apharmaceutically acceptable salt thereof. In some embodiments theantibiotic is not ampicillin or a pharmaceutically acceptable saltthereof.

In some embodiments the antibiotic is a quinolone, fluoroquinolone, or apharmaceutically acceptable salt thereof. In some embodiments, theantibiotic is ciprofloxacin, besifloxacin, enoxacin, nalidixic acid,norfloxacin, levofloxacin, moxifloxacin, pefloxin, a combinationthereof, or a pharmaceutically acceptable salt thereof.

In some embodiments the antibiotic is not a quinolone, fluoroquinolone,or a pharmaceutically acceptable salt thereof. In some embodiments theantibiotic is not ciprofloxacin or a pharmaceutically acceptable saltthereof.

In some embodiments the antibiotic is a cephalosporin or apharmaceutically acceptable salt thereof. In some embodiments theantibiotic is ceftriaxone, cefoperazone, a combination thereof, or apharmaceutically acceptable salt thereof.

In some embodiments the antibiotic is not a cephalosporin or apharmaceutically acceptable salt thereof.

In some embodiments the antibiotic is a peptide, glycopeptide, or apharmaceutically acceptable salt thereof. In some embodiments, theantibiotic is vancomycin, polymyxin B, a combination thereof, or apharmaceutically acceptable salt thereof. In some embodiments, theantibiotic is a cathelicidin peptide.

In some embodiments the antibiotic is not a peptide, glycopeptide, or apharmaceutically acceptable salt thereof. In some embodiments, theantibiotic is not a cathelicidin peptide.

In some embodiments, the antibiotic is chloramphenicol, dirithromycin,erythromycin, doxycycline, tetracycline, linezolid, bacitracin,fosfomycin, fosmidomycin, ampicillin, amoxicillin, cloxacillin,piperacillin, oxacillin, ceftriaxone, cefoperazone, vancomycin,polymyxin B, ciprofloxacin, besifloxacin, enoxacin, nalidixic acid,norfloxacin, levofloxacin, moxifloxacin, pefloxin, novobiocin,pentamidine, rifampicin, trimethoprim, sulfamethoxazole, a combinationthereof, or a pharmaceutically acceptable salt thereof.

In some embodiments, the antibiotic is apramycin, neomycin, paromycin,spectinomycin, chloramphenicol, dirithromycin, erythromycin,doxycycline, tetracycline, linezolid, bacitracin, fosfomycin,fosmidomycin, ampicillin, amoxicillin, cloxacillin, a combinationthereof, or a pharmaceutically acceptable salt thereof.

In some embodiments, the antibiotic is erythromycin, dirithromycin,levofloxacin, norfloxacin, ciprofloxacin, enoxacin, moxifloxacin,besifloxacin polymyxin B, a combination thereof, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the antibiotic is cefoperazone, novobiocin,ampicillin, cloxacillin, oxacillin, doxycycline or a pharmaceuticallyacceptable salt thereof.

In some embodiments, an antibiotic agent is Amikacin, Apramycin,Gentamicin, Kanamycin, Neomycin, Tobramycin, Paromomycin, Streptomycin,Spectinomycin, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem,Cefadroxil, Cefazolin, Cefalothin, Cefalexin, Cefaclor, Cefamandole,Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren,Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten,Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole,Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin,Lipopeptide, Daptomycin, Azithromycin, Clarithromycin, Dirithromycin,Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin,Aztreonam, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin,Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin,Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin,Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin,Ticarcillin, Bacitracin, Colistin, Polymyxin B, Besifloxacin,Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Pefloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin,Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine,Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide,Sulfasalazine, Sulfisoxazole, Sulfonamidochrysoidine, Demeclocycline,Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Arsphenamine,Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin,Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline,Tinidazole, Trimethoprim, Clofazimine, Dapsone, Capreomycin,Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide,Rifampicin, Rifabutin, Rifapentine or Streptomycin, or apharmaceutically acceptable salt thereof.

In some embodiments, the antibiotic agent causes a decrease in the pHgradient across the microorganism's cytoplasmic membrane.

In some embodiments, the antibiotic agent causes an increase in themicroorganism's cytoplasmic membrane potential.

In some embodiments, the antibiotic agent is a cationic antibioticagent, an antibiotic agent that is an energy dependent efflux substrate,an antibiotic agent for which entry depends on membrane potential, or anantibiotic agent that disrupts membrane potential as a primary mechanismof action.

Also provided herein are methods for treating a bacterial infection, ora disease, disorder or condition arising from a bacterial infection,comprising administering to a subject in need thereof an effectiveamount of (i) bicarbonate and (ii) an antibiotic.

Innate Immunity Factors

In some embodiments, the antimicrobial agent is an innate immunityfactor.

Innate immunity factors are components of the innate immune systemincluding several classes of soluble molecules present in blood,extracellular fluid and epithelial secretions that are intrinsicallybiologically active and affect pathogen survival. Examples includeantimicrobial peptides such as defensins, antimicrobial enzymes, such aslysozyme and antimicrobial secretions, such as hyaluronic acid.

Accordingly, in some embodiments, an antimicrobial agent is an innateimmunity factor such as an antimicrobial peptide, an antimicrobialenzyme, or an antimicrobial secretion. In some embodiments, the innateimmunity factor is LL-37, indolicidin, bactenesin, defensin,alpha-defensin, a bile salt, lysozyme, protegrin, or hyaluronic acid, ora pharmaceutically acceptable salt thereof. In some embodiments, theinnate immunity factor is a cathelicidin peptide. In some embodiments,the innate immunity factor is not a cathelicidin peptide. In someembodiments, the innate immunity factor is not LL-37.

Also provided herein are methods for treating a microbial infection, ora disease, disorder or condition arising from a microbial infection,comprising administering to a subject in need thereof an effectiveamount of (i) bicarbonate and (ii) an innate immunity factor.

Antiviral Agents

In some embodiments, the antimicrobial agent is an antiviral agent.

In some embodiments, an antiviral agent is Abacavir, Acyclovir,Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir,Atripla, Balavir, Cidofovir, Combivir, Dolutegravir, Darunavir,Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine,Enfuvirtide, Entecavir, Ecoliever, Famciclovir, Fomivirsen,Fosamprenavir, Foscarnet, Fosfonet, Fusion inhibitor, Ganciclovir,Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine,Integrase inhibitor, Interferon type III, Interferon type II, Interferontype I, Interferon, Lamivudine, Lopinavir, Loviride, Maraviroc,Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, Nitazoxanide,Nucleoside analogues, Norvir, Oseltamivir, Peginterferon alfa-2a,Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Protease inhibitor,Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rimantadine,Ritonavir, Pyramidine, Saquinavir, Sofosbuvir, Stavudine, Synergisticenhancer (antiretroviral), Telaprevir, Tenofovir, Tenofovir disoproxil,Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir,Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine,Zanamivir or Zidovudine, or a pharmaceutically acceptable salt thereof.

Also provided herein are methods for treating a viral infection, or adisease, disorder or condition arising from a viral infection,comprising administering to a subject in need thereof an effectiveamount of (i) bicarbonate and (ii) an antiviral agent.

Anti-Fungal Agents

In some embodiments, the antimicrobial agent is an anti-fungal agent.

In some embodiments, an anti-fungal agent is Amphotericin B, Candicidin,Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Bifonazole,Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole,Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole,Sertaconazole, Sulconazole, Tioconazole, Albaconazole, Efinaconazole,Epoxiconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole,Propiconazole, Ravuconazole, Terconazole, Voriconazole, Abafungin,Amorolfin, Butenafine, Naftifine, and Terbinafine, Anidulafungin,Caspofungin, Micafungin, Aurones, Benzoic acid, Ciclopirox, Flucytosine,Griseofulvin, Haloprogin, Tolnaftate, Undecylenic acid, Crystal violet,Orotomide or Miltefosine, or a pharmaceutically acceptable salt thereof.

Also provided herein are methods for treating a fungal infection, or adisease, disorder or condition arising from a fungal infection,comprising administering to a subject in need thereof an effectiveamount of (i) bicarbonate and (ii) an anti-fungal agent.

Anti-Parasitic Agents

In some embodiments, the antimicrobial agent is an anti-parasitic agent.

In some embodiments, an anti-parasitic agent is Nitazoxanide,Melarsoprol, Eflornithine, Metronidazole, Tinidazole, Miltefosine,Ancylostoma caninum, Mebendazole, Pyrantel pamoate, Thiabendazole,Diethylcarbamazine, Ivermectin, Niclosamide, Praziquantel, Albendazole,Antitrematodes, Praziquantel, Rifampin, Amphotericin B or Fumagillin, ora pharmaceutically acceptable salt thereof.

Also provided herein are methods for treating a parasitic infection, ora disease, disorder or condition arising from a parasitic infection,comprising administering to a subject in need thereof an effectiveamount of (i) bicarbonate and (ii) an anti-parasitic agent.

Pentamidine and Analogs Thereof

In some embodiments, an antimicrobial agent is diamidine or apharmaceutically acceptable salt thereof. In some embodiments, anantimicrobial agent is propamidine or a pharmaceutically acceptable saltthereof. In some embodiments, an antimicrobial agent is pentamidine or apharmaceutically acceptable salt thereof. In some embodiments, anantimicrobial agent is not a diamidine, or a pharmaceutically acceptablesalt thereof. In one embodiment, an antimicrobial agent is notpentamidine, or a pharmaceutically acceptable salt thereof.

Pentamidines include analogs thereof, including pharmaceuticallyacceptable salts and solvates thereof. Various analogs of pentamidineare known in the art. In some embodiments, the analogs of pentamidinecan have an alkylene linker that ranges from 2 to 10 carbon atoms.

Pentamidine is provided in Formula I:

In some embodiments the analogs of pentamidine are compounds of FormulaII or a pharmaceutically acceptable salt thereof.

wherein n is 0, 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is 1.In some embodiments, n is an integer from 2 to 4.Microorganisms

The methods provided herein comprise contacting a microorganism with anantimicrobial agent in the presence of bicarbonate. In some embodiments,the presence of bicarbonate modulates the microorganism's growth inresponse to the antimicrobial agent.

In some embodiments, the presence of bicarbonate decreases themicroorganism's growth in response to antimicrobial agent. In someembodiments, the presence of bicarbonate decreases the microorganism'sgrowth in response to the antimicrobial agent by at least 10-fold. Insome embodiments, the presence of bicarbonate decreases themicroorganism's growth in response to the antimicrobial agent by atleast 20-fold. In some embodiments, the presence of bicarbonatedecreases the microorganism's growth in response to the antimicrobialagent by at least 30-fold.

In some embodiments, the presence of bicarbonate increases themicroorganism's growth response to the antimicrobial agent. In someembodiments, the presence of bicarbonate increases the microorganism'sgrowth in response to the antimicrobial agent by at least 10-fold.

In some embodiments, a method described herein inhibits the growth of amicroorganism.

In some embodiments of the methods described herein, a microorganism ispresent on a substrate. In some examples, a substrate is glass, metal,plastic, latex, ceramic, cement, wood, grout or stone.

In some embodiments, a subject is infected with the microorganism. Insome embodiments, the subject is a mammal. In some embodiments, thesubject is a human. In some embodiments, the subject has a microbialinfection. In some embodiments, the microbial infection is a pulmonary,lung, otic, oral, nasal, sinus, ophthalmic, intraocular, dermal,cardiovascular, kidney, urinary, gastrointestinal, rectal, vaginal orneurological infection.

In some embodiments of the methods described herein, a microorganism isa bacterium. In some embodiments, a bacterium is a Gram negativebacterium. In some embodiments, a bacterium is a Gram positivebacterium. In some embodiments, a bacterium is a spiral-shapedbacterium, a filamentous bacterium, a pleomorphic bacterium or arectangular bacterium. In other embodiments, a bacterium is asphere-shaped bacterium, a rod-shaped bacterium, a spiral-shapedbacterium, a filamentous bacterium, a pleomorphic bacterium or arectangular bacterium. In one embodiment, a bacterium is a Gram positiverod-shaped bacterium. In another embodiment, a bacterium is a Gramnegative rod-shaped bacterium. In yet another embodiment, a bacterium isa Gram positive sphere-shaped bacterium. In a further embodiment, abacterium is a Gram negative sphere-shaped bacterium.

In some aspects of the methods described herein, a bacterium is anobligate aerobe or an obligate anaerobe. In other aspects of the methodsdescribed herein, a bacterium is a facultative anaerobe.

In some embodiments of the methods described herein, a bacterium is aspecies of Acinetobacter, Actinomyces, Aerococcus, Agrobacterium,Anaplasma, Azorhizobium, Azotobacter, Bacillus, Bacteroides, Bartonella,Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium,Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium,Coxiella, Ehrlichia, Enterobacter, Enterococcus, Escherichia,Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter,Klebsiella, Lactobacillus, Lactococcus, Legionella, Listeria,Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium,Mycoplasma, Neisseria, Pasteurella, Pediococcus, Peptostreptococcus,Porphyromonas, Prevotella, Pseudomonas, Rhizobium, Rickettsia,Rochalimaea, Rothia, Salmonella, Serratia, Shigella, Sarcina, Spirillum,Spirochaetes, Staphylococcus, Stenotrophomonas, Streptobacillus,Streptococcus, Tetragenococcus, Treponema, Vibrio, Viridans, Wolbachiaor Yersinia.

In some embodiments of the methods described herein, a bacterium isAcetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii,Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasmaphagocytophilum, Azorhizobium caulinodans, Azotobacter vinelandii,Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillusfusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillusmycoides, Bacillus stearothermophilus, Bacillus subtilis, BacillusThuringiensis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroidesmelaninogenicus, Bartonella henselae, Bartonella Quintana, Bordetellabronchiseptica, Bordetella pertussis, Borrelia burgdorferi. Brucellaabortus, Brucella melitensis, Brucella suis, Burkholderia mallei,Burkholderia pseudomallei, Burkholderia cepacia, Calymmatobacteriumgranulomatis, Campylobacter coli, Campylobacter fetus, Campylobacterjejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophilapneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridiumdifficile, Clostridium perfringens, Clostridium tetani, Corynebacteriumdiphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Ehrlichiachaffeensis, Enterobacter cloacae, Enterococcus avium, Enterococcusdurans, Enterococcus faecalis, Enterococcus faecium, Enterococcusgalllinarum, Enterococcus maloratus, Escherichia coli, Francisellatularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilusducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophiluspertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiellapneumoniae, Lactobacillus acidophilus, Lactobacillus bulgaricus,Lactobacillus casei, Lactococcus lactis, Legionella pneumophila,Listeria monocytogenes, Methanobacterium extroquens, Microbacteriummultiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacteriumavium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacteriumintracellulare, Mycobacterium leprae, Mycobacterium lepraemurium,Mycobacterium phlei, Mycobacterium smegmatis, Mycobacteriumtuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasmahominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseriagonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurellatularensis, Peptostreptococcus, Porphyromonas gingivalis, Prevotellamelaninogenica, Pseudomonas aeruginosa, Rhizobium radiobacter,Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana,Rickettsia rickettsii, Rickettsia trachomae, Rochalimaea henselae,Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis,Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigelladysenteriae, Spirillum volutans, Staphylococcus aureus, Staphylococcusepidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae,Streptococcus avium, Streptococcus bovis, Streptococcus cricetus,Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus,Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior,Streptococcus mitis, Streptococcus mutans, Streptococcus oralis,Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus,Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus,Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma,Vibrio parahaemolyticus, Vibrio vulnificus, Viridans streptococci,Wolbachia, Yersinia enterocolitica, Yersinia pestis or Yersiniapseudotuberculosis.

In some embodiments of the methods described herein, the bacterium isEscherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae,Pseudomonas aeruginosa, Burkhloderia cenocepacia, Burkhloderiamultivorans, Enterococcus faecalis or Staphylococcus aureus. In someembodiments, the bacterium is Escherichia coli. In some embodiments, thebacterium is Staphylococcus aureus. In some embodiments of the methodsdescribed herein, the bacterium is methicillin-resistant Staphylococcusaureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA).

In some embodiments of the methods described herein, the bacterium isnot Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae,Pseudomonas aeruginosa, Burkhloderia cenocepacia, Burkhloderiamultivorans, Enterococcus faecalis or Staphylococcus aureus. In someembodiments, the bacterium is not Escherichia coli. In some embodiments,the bacterium is not Staphylococcus aureus. In some embodiments of themethods described herein, the bacterium is not methicillin-resistantStaphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcusaureus (MSSA).

In some embodiments, a bacterial infection is an infection byEscherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae,Pseudomonas aeruginosa, Burkhloderia cenocepacia, Burkhloderiamultivorans, Enterococcus faecalis or Staphylococcus aureus (e.g., MRSAor MSSA).

In some embodiments, the microbial infection treated by the methods andcompositions described herein is a bacterial infection and theantimicrobial agent is an antibiotic. In some embodiments, theantimicrobial agent is an antibiotic, anti-fungal agent, or innateimmunity factor. In some embodiments, the antibiotic is a macrolide,tetracycline, peptide, glycopeptide, penicillin, cephalosporin,quinolone, or fluoroquinolone. In some embodiments, the antibiotic ispentamidine, erythromycin, dirithromycin, levofloxacin, norfloxacin,ciprofloxacin, enoxacin, moxifloxacin, besifloxacin or polymyxin B, or apharmaceutically acceptable salt thereof.

In some embodiments of the methods described herein, a microorganism isa virus. In some examples, a virus is an Adenoviridae, Herpesviridae,Poxviridae, Papillomaviridae, Polyomaviridae, Parvoviridae, Reoviridae,Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Hepeviridae,Flaviviridae, Togaviridae, Arenaviridae, Bunyaviridae, Filoviridae,Paramyxoviridae, Rhabdoviridae, Orthomyxoviridae, Retroviridae orHepadnaviridae family member. In some embodiments, a virus isAdenovirus, Herpes simplex virus type 1, Herpes simplex virus type 2,Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Humanherpesvirus type 8, Smallpox, Human papillomavirus, BK virus, JC virus,Parvovirus B19, Rotavirus, Orbivirus, Coltivirus, Banna virus, Humanastrovirus, Norwalk virus, coxsackievirus, Hepatitis A virus, HepatitisB virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus,poliovirus, rhinovirus, Severe acute respiratory syndrome virus, yellowfever virus, dengue virus, West Nile virus, TBE virus, Rubella virus,Lassa virus, Crimean-Congo hemorrhagic fever virus, Hantaan virus, Ebolavirus, Marburg virus, Measles virus, Mumps virus, Parainfluenza virus,Respiratory syncytial virus, Rabies virus, Influenza virus or Humanimmunodeficiency virus (HIV).

In some embodiments, the microbial infection treated by the methods andcompositions described herein is a viral infection and the antimicrobialagent is an antiviral agent.

In some embodiments of the methods provided herein, a microorganism is afungus. In some embodiments, a fungus is a yeast. In an illustrativeembodiment, the fungus is Pneumocystis carinii. In some aspects, methodsare provided to treat or prevent Pneumocystis carinii pneumonia,comprising administering to a subject in need thereof an effectiveamount of (i) bicarbonate and (ii) an antimicrobial agent. In someembodiments, the microbial infection treated by the methods andcompositions described herein is a fungal or yeast infection and theantimicrobial agent is an anti-fungal agent. In some embodiments, thefungal infection is a Pneumocystis carinii infection and the disease,disorder or condition arising from the fungal infection is Pneumocystiscarinii pneumonia.

In some embodiments of the methods disclosed herein, a fungus is anAgaricus species, Amanita species, Armillaria species, Aspergillusspecies, Boletus species, Caloplaca species, Candida species, Cladoniaspecies, Coprinellus species, Coprinopsis species, Cortinarius species,Cyathus species, Deadly fungus species, Entoloma species, Fusariumspecies, Gymnopilus species, Gymnopus species, Hebeloma species,Hygrocybe species, Hygrophorus species, Inocybe species, Lactariusspecies, Lactifluus species, Lecanora species, Lepiota species,Leucoagaricus species, Lichen species of Montana, Leccinum species,Marasmius species, Pleurotus species, Mycosphaerella species, Panaeolusspecies, Penicillium species, Peniophora species, Pertusaria species,Phaeocollybia species, Pholiota species, Pholiotina species, Pluteusspecies, Poisonous fungus species, Psathyrella species, Psilocybespecies, Psilocybin mushroom species, Puccinia species, Russula species,Scleroderma species, Serpula species, Trametes species, Tricholomaspecies, Tuber species, or Tulostoma species.

In some embodiments of the methods disclosed herein, a microorganism isa parasite. In some embodiments, a parasite is a protozoan.

In some embodiments of the methods provided herein, a parasite is anAcanthamoeba species, Balamuthia mandrillaris, B. divergens, B.bigemina, B. equi, B. microfti, B. duncani, Balantidium coli, aBlastocystis species, a Cryptosporidium species, Cyclosporacayetanensis, Dientamoeba fragilis, Entamoeba histolytica, Giardialamblia, Isospora belli, a Leishmania species, Naegleria fowleri,Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale curtisi,Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium knowlesi,Rhinosporidium seeberi, Sarcocystis bovihominis, Sarcocystis suihominis,Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei,Trypanosoma cruzi, Cestoda, Taenia multiceps, Diphyllobothrium latum,Echinococcus granulosus, Echinococcus multilocularis, E. vogeli, E.oligarthrus, Hymenolepis nana, Hymenolepis diminuta, Taenia saginata,Taenia solium, Bertiella mucronata, Bertiella studeri, Spirometraerinaceieuropaei, Echinostoma echinatum, Schistosoma mekongi,Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis,Clonorchis sinensis; Clonorchis viverrini, Fasciolopsis buski,Schistosoma mansoni, Schistosoma intercalatum, Dicrocoelium dendriticum,Fasciola hepatica, Fasciola gigantica, Metagonimus yokogawai, Metorchisconjunctus, Paragonimus westermani, Paragonimus africanus, Paragonimuscaliensis, Paragonimus kellicotti, Paragonimus skrjabini, Paragonimusuterobilateralis, Schistosoma japonicum, a Schistosoma species,Trichobilharzia regenti, Schistosomatidae, Schistosoma haematobium,Ancylostoma duodenale, Necator americanus, Angiostrongyluscostaricensis, an Anisakis Ascaris species, Ascaris lumbricoides,Baylisascaris procyonis, Brugia malayi, Brugia timori, Dioctophymerenale, Dracunculus medinensis, Enterobius vermicularis, Enterobiusgregorii, Gnathostoma spinigerum, Gnathostoma hispidum, Halicephalobusgingivali, Loa loa filarial, Mansonella streptocerca, Onchocercavolvulus, Strongyloides stercoralis, Thelazia californiensis, Thelaziacallipaeda, Toxocara canis, Toxocara cati, Trichinella spiralis,Trichinella britovi, Trichinella nelsoni, Trichinella native, Trichuristrichiura, Trichuris vulpis, Wuchereria bancrofti, Archiacanthocephala,Moniliformis moniliformis, Linguatula serrate, Oestroidea,Calliphoridae, Sarcophagidae, Cochliomyia hominivorax, Tunga penetrans,Dermatobia hominis, Pediculus humanus capitis, Pediculus humanushumanus, Pthirus pubis, Demodex folliculorum/brevis/canis, Sarcoptesscabiei, an Arachnida class member, Trombiculidae, Pulex irritans,Cimicidae, Cimex lectularius, an Ixodidae family member or an Argasidaefamily member.

In another aspect, provided herein are methods of treating a microbialinfection, or a disease, disorder or condition arising from a microbialinfection, comprising administering, to a subject in need thereof, aneffective amount of (i) bicarbonate and (ii) an antimicrobial agent,wherein the antimicrobial agent is an antimicrobial agent whoseconcentration in a cell of the microorganism is increased by one or bothof (1) decrease in pH gradient across the microorganism's cytoplasmicmembrane; and (2) increase in the microorganism's cytoplasmic membranepotential and wherein the antimicrobial agent is not an aminoglycoside.

In another aspect, provided herein are methods of treating a microbialinfection, or a disease, disorder or condition arising from a microbialinfection, comprising administering to a subject in need thereof aneffective amount of (i) bicarbonate and (ii) an antimicrobial agent,wherein the microbial infection is caused by a microorganism, andwherein the antimicrobial agent is a cationic antimicrobial agent,antimicrobial agent that is an energy dependent efflux substrate,antimicrobial agent for which entry depends on membrane potential, or anantimicrobial agent that disrupts membrane potential as a primarymechanism of action.

Bicarbonate

Bicarbonate forms the dominant buffering system in the human body, whichplays an important role in maintaining the pH of blood around 7.4.

The methods provided herein comprise contacting a microorganism with anantimicrobial agent in the presence of bicarbonate.

In some embodiments, the bicarbonate potentiates antimicrobial activityby increasing the effective intracellular levels of variousantimicrobial agents or enhancing their ability to collapse PMF.

In some embodiments, the bicarbonate is potassium, lithium, calcium,magnesium, sodium, ammonium or zinc bicarbonate. In some embodiments,the bicarbonate is sodium bicarbonate or ammonium bicarbonate. In someembodiments, the bicarbonate is sodium bicarbonate.

In some embodiments, the dosage or amount of the bicarbonate can be anamount to provide physiological concentrations of bicarbonate, or about25 mM of bicarbonate. In some embodiments, the bicarbonate can beprovided in a composition comprising about 1 mM to about 150 mMbicarbonate, or about 20 mM to about 50 mM bicarbonate. In someembodiments, the bicarbonate can be provided in a composition comprisingabout 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140mM, 145 mM, or even about 150 mM bicarbonate. In some embodiments, thebicarbonate can be provided in a composition comprising about 0.01 wt %to about 1.0 wt % bicarbonate, or about 0.20 wt % to about 0.5 wt %bicarbonate. In some embodiments, the effective amount or dosage ofbicarbonate can be about 0.01 mg to about 1 mg per kg of body weight ofthe subject. In some embodiments, the bicarbonate can be provided in acomposition comprising about 0.01 mg to about 1 mg of bicarbonate. Insome embodiments, the amount of the bicarbonate is a physiologicalconcentration of the bicarbonate. In some embodiments, the bicarbonateis present in a composition having a concentration of about 1 mM toabout 150 mM of bicarbonate. In some aspects, the bicarbonate is presentin a composition being about 0.01 wt % to about 1 wt % of bicarbonate

In some embodiments, bicarbonate useful in the methods and compositionsdescribed herein is a component of a buffer. As with any buffer system,bicarbonate acts as a buffer in solution at neutral pH due to thepresence of both a weak acid (carbonic acid; H₂CO₃) and its conjugatebase (HCO₃ ⁻) where these species have the capacity to buffer the addedbase or acid, respectively. As shown below, in a bicarbonate buffersystem, dissolved carbon dioxide and bicarbonate ion are at equilibrium.CO₂+H₂O⇄H₂CO₃⇄HCO₃ ⁻+H⁺The pKa of carbonate is 6.1 and thus at neutral pH typically used inmicrobiological testing or that of the human body (e.g., pH 7.4), thevast majority of the carbonate-bicarbonate buffer system is present asthe conjugate base bicarbonate ion. Thus, the active species is likelythe bicarbonate ion, consistent with its ability to buffer protons thatfigure in the proton gradient of the proton motive force. Althoughcarbonate is present in this system, the system may be described as abicarbonate buffer because bicarbonate is the dominant species.Methods of Use

The invention provides methods for inhibiting the growth of amicroorganism, comprising contacting the microorganism with an effectiveamount of (i) bicarbonate and (ii) an antimicrobial agent. In someexamples, an antimicrobial agent is pentamidine, an antibiotic agent, aninnate immunity factor, an antiviral agent, an anti-fungal agent or ananti-parasitic agent.

In some embodiments, invention provides methods for inhibiting thegrowth of a virus, a bacterium, a fungus or a parasite, comprisingcontacting the virus, the bacterium, the fungus or the parasite with aneffective amount of (i) bicarbonate and (ii) an antimicrobial agent;

wherein the bacterium is:

(a) a spiral-shaped bacterium, a filamentous bacterium, a pleomorphicbacterium or a rectangular bacterium;

(b) an obligate aerobe or an obligate anaerobe;

(c) a Gram positive rod-shaped bacterium;

(d) a Gram negative sphere-shaped bacterium;

(e) a species of Acinetobacter, Actinomyces, Aerococcus, Agrobacterium,Anaplasma, Azorhizobium, Azotobacter, Bacillus, Bacteroides, Bartonella,Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium,Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium,Coxiella, Ehrlichia, Enterobacter, Enterococcus, Francisella,Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella,Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium,Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma,Neisseria, Pasteurella, Pediococcus, Peptostreptococcus, Porphyromonas,Prevotella, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia,Salmonella, Serratia, Shigella, Sarcina, Spirillum, Spirochaetes,Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus,Treponema, Vibrio, Viridans, Wolbachia or Yersinia; or

f) Staphylococcus epidermidis.

Provided herein are methods for inhibiting the growth of Staphylococcusaureus, comprising contacting the Staphylococcus aureus with aneffective amount of (i) bicarbonate and (ii) pentamidine or apharmaceutically acceptable salt thereof.

Additionally provided herein are methods for treating or preventing amicrobial infection, comprising administering to a subject in needthereof an effective amount of (i) bicarbonate and (ii) an antimicrobialagent;

wherein the microbial infection is an infection by a virus, a bacterium,a fungus or a parasite; and

wherein the bacterium is:

(a) a spiral-shaped bacterium, a filamentous bacterium, a pleomorphicbacterium or a rectangular bacterium;

(b) an obligate aerobe or an obligate anaerobe;

(c) a Gram positive rod-shaped bacterium;

(d) a Gram negative sphere-shaped bacterium;

(e) a species of Acinetobacter, Actinomyces, Aerococcus, Agrobacterium,Anaplasma, Azorhizobium, Azotobacter, Bacillus, Bacteroides, Bartonella,Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium,Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium,Coxiella, Ehrlichia, Enterobacter, Enterococcus, Francisella,Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella,Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium,Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma,Neisseria, Pasteurella, Pediococcus, Peptostreptococcus, Porphyromonas,Prevotella, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia,Salmonella, Serratia, Shigella, Sarcina, Spirillum, Spirochaetes,Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus,Treponema, Vibrio, Viridans, Wolbachia or Yersinia; or

(f) Staphylococcus epidermidis.

In some embodiments, disclosed herein is a method for inhibiting thegrowth of a virus, a bacterium, a fungus or a parasite, comprisingcontacting the virus, the bacterium, the fungus or the parasite with aneffective amount of (i) bicarbonate and (ii) an antimicrobial agent;

wherein the antimicrobial agent is an antiviral agent, an anti-fungalagent, an anti-parasitic agent, an antibiotic agent, or an innateimmunity factor; and

wherein the antibiotic agent is

(a) a macrolide, a tetracycline, a cephalosporin, a quinolone, arifampin or a fluoroquinolone; or

(b) Amoxicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin,Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin,Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin orTicarcillin, or a pharmaceutically acceptable salt thereof.

In other embodiments, disclosed herein is a method of treating orpreventing a microbial infection, comprising administering to a subjectin need thereof an effective amount of (i) bicarbonate and (ii) anantimicrobial agent;

wherein the microbial infection is an infection by a virus, a bacterium,a fungus or a parasite;

wherein the antimicrobial agent is an antiviral agent, an anti-fungalagent, an anti-parasitic agent, an antibiotic agent, or an innateimmunity factor; and

wherein the antibiotic agent is

(a) a macrolide, a tetracycline, a cephalosporin, a quinolone, arifampin or a fluoroquinolone; or

(b) Amoxicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin,Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin,Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin orTicarcillin, or a pharmaceutically acceptable salt thereof.

In other embodiments, disclosed herein is a method of treating orpreventing a microbial infection, comprising administering to a subjectin need thereof an effective amount of (i) bicarbonate and (ii) anantimicrobial agent;

wherein the microbial infection is an infection by a bacterium, andwherein the bacterium is Acetobacter aurantius, Acinetobacter baumannii,Actinomyces israelii, Agrobacterium radiobacter, Agrobacteriumtumefaciens, Anaplasma phagocytophilum, Azorhizobium caulinodans,Azotobacter vinelandii, Bacillus anthracis, Bacillus brevis, Bacilluscereus, Bacillus fusiformis, Bacillus licheniformis, Bacillusmegaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillussubtilis, Bacillus Thuringiensis, Bacteroides fragilis, Bacteroidesgingivalis, Bacteroides melaninogenicus, Bartonella henselae, BartonellaQuintana, Bordetella bronchiseptica, Bordetella pertussis, Borreliaburgdorferi. Brucella abortus, Brucella melitensis, Brucella suis,Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia,Calymmatobacterium granulomatis, Campylobacter coli, Campylobacterfetus, Campylobacter jejuni, Campylobacter pylori, Chlamydiatrachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci,Clostridium botulinum, Clostridium difficile, Clostridium perfringens,Clostridium tetani, Corynebacterium diphtheriae, Corynebacteriumfusiforme, Coxiella burnetii, Ehrlichia chaffeensis, Enterobactercloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis,Enterococcus faecium, Enterococcus galllinarum, Enterococcus maloratus,Escherichia coli, Francisella tularensis, Fusobacterium nucleatum,Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae,Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilusvaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillusacidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactococcuslactis, Legionella pneumophila, Listeria monocytogenes, Methanobacteriumextroquens, Microbacterium multiforme, Micrococcus luteus, Moraxellacatarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacteriumdiphtheriae, Mycobacterium intracellulare, Mycobacterium leprae,Mycobacterium lepraemurium, Mycobacterium phlei, Mycobacteriumsmegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasmagenitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasmapneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurellamultocida, Pasteurella tularensis, Peptostreptococcus, Porphyromonasgingivalis, Prevotella melaninogenica, Pseudomonas aeruginosa, Rhizobiumradiobacter, Rickettsia prowazekii, Rickettsia psittaci, Rickettsiaquintana, Rickettsia rickettsii, Rickettsia trachomae, Rochalimaeahenselae, Rochalimaea quintana, Rothia dentocariosa, Salmonellaenteritidis, Salmonella typhi, Salmonella typhimurium, Serratiamarcescens, Shigella dysenteriae, Spirillum volutans, Staphylococcusaureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia,Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis,Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis,Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis,Streptococcus mitior, Streptococcus mitis, Streptococcus mutans,Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis,Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibriocholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus,Viridans streptococci, Wolbachia, Yersinia enterocolitica, Yersiniapestis or Yersinia pseudotuberculosis.

Provided herein are methods of modulating a microorganism's response toan antimicrobial agent. The methods comprise contacting a microorganismwith an antimicrobial agent in the presence of bicarbonate, whereby thebicarbonate modulates the microorganism's response to the antimicrobialagent. In some embodiments, the bicarbonate potentiates themicroorganism's response to the antimicrobial agent, resulting in anincreased response. In some embodiments, the bicarbonate lessens themicroorganism's response to the antimicrobial agent, resulting in adecreased response. In some embodiments, the antimicrobial agent is aninnate immunity factor or a macrolide, tetracycline, peptide,glycopeptide, penicillin, cephalosporin, quinolone, rifampin,fluoroquinolone, an antifungal agent or aminoglycoside antibiotic. Insome embodiments, the antimicrobial agent is not an aminoglycoside.

In some embodiments, the presence of bicarbonate decreases themicroorganism's growth in response to the antimicrobial agent. In someembodiments, the presence of bicarbonate decreases the microorganism'sgrowth in response to the antimicrobial agent by at least 2-fold,4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, or 30-fold.

In some embodiments, the presence of bicarbonate increases themicroorganism's growth response to the antimicrobial agent. In someembodiments, the presence of bicarbonate increases the microorganism'sgrowth in response to the antimicrobial agent by at least 2-fold,4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, or 30-fold.

In some embodiments, the presence of bicarbonate changes the MIC of theantimicrobial agent by at least 2-fold, 4-fold, 5-fold, 8-fold, 10-fold,15-fold, 20-fold, or 30-fold.

Also provided herein are methods for treating a microbial infection, ora disease, disorder or condition arising from a microbial infection. Themethods comprise administering to a subject in need thereof an effectiveamount of (i) bicarbonate and (ii) an antimicrobial agent, wherein theantimicrobial agent targets the membrane potential of a cell and/or issubject to an increase in intracellular concentration by one or both of(1) decrease in pH gradient across the microorganism's cytoplasmicmembrane; and (2) increase in the microorganism's cytoplasmic membranepotential. In some embodiments, the antimicrobial agent is a macrolide,tetracycline, peptide, glycopeptide, penicillin, cephalosporin,quinolone, fluoroquinolone, rifampin, innate immunity factor, orcombinations thereof. In some embodiments, the antimicrobial agent isnot an aminoglycoside.

In one embodiment, disclosed herein is a method of treating orpreventing an Staphylococcus aureus infection, comprising administeringto a subject in need thereof an effective amount of (i) bicarbonate and(ii) pentamidine or a pharmaceutically acceptable salt thereof.

In some embodiments, the methods described herein comprise administeringto a subject in need thereof an effective amount of (i) bicarbonate and(ii) indolicidin, bactenesin, defensin, alpha-defensin, a bile salt,lysozyme, protegrin, or hyaluronic acid.

The present invention provides methods for potentiating an antimicrobialagent comprising administering to a subject in need thereof an effectiveamount of (i) bicarbonate and (ii) the antimicrobial agent, wherein theantimicrobial agent is an antimicrobial agent whose concentration in acell of the microorganism is increased by one or both of (1) decrease inpH gradient across the microorganism's cytoplasmic membrane; and (2)increase in the microorganism's cytoplasmic membrane potential. In someembodiments, the antimicrobial agent is a macrolide, tetracycline,peptide, glycopeptide, penicillin, cephalosporin, quinolone,fluoroquinolone, rifampin, innate immunity factor, or combinationsthereof. In some embodiments, the antimicrobial agent is not anaminoglycoside.

Also provided herein is a use of bicarbonate and an antimicrobial agentto treat a microbial infection, or a disease, disorder or conditionarising from a microbial infection. In one embodiment, the inventionprovides a use of bicarbonate and an antimicrobial agent for thepreparation of a medicament to treat a microbial infection, or adisease, disorder or condition arising from a microbial infection,wherein the antimicrobial agent is an antimicrobial agent whoseconcentration in a cell of the microorganism is increased by one or bothof (1) decrease in pH gradient across the microorganism's cytoplasmicmembrane; and (2) increase in the microorganism's cytoplasmic membranepotential. In some embodiments, the antimicrobial agent is anaminoglycoside, macrolide, tetracycline, peptide, glycopeptide,penicillin, cephalosporin, quinolone, fluoroquinolone, rifampin, innateimmunity factor, or combinations thereof. In some embodiments, theantimicrobial agent is not an aminoglycoside.

In some embodiments, the methods described herein may spare the naturalor beneficial microbiota of a host.

In one embodiment, the invention provides bicarbonate and anantimicrobial agent for use in therapy. In another embodiment, theinvention provides bicarbonate and an antimicrobial agent for use in amethod of treating a microbial infection, or a disease, disorder orcondition arising from a microbial infection.

Also included is bicarbonate for use with an antimicrobial agent totreat a microbial infection, or a disease, disorder or condition arisingfrom a microbial infection. Further provided is bicarbonate for use withan antimicrobial agent to treat a microbial infection, or a disease,disorder or condition arising from a microbial infection, wherein theantimicrobial agent's concentration in a cell of the microorganism isincreased by one or both of (1) decrease in pH gradient across themicroorganism's cytoplasmic membrane; and (2) increase in themicroorganism's cytoplasmic membrane potential. In some embodiments, theantimicrobial agent is a macrolide, tetracycline, peptide, glycopeptide,penicillin, cephalosporin, quinolone, fluoroquinolone, rifampin, innateimmunity factor, or combinations thereof. In some embodiments, theantimicrobial agent is not an aminoglycoside.

Provided herein are methods of treating a topical microbial infection,or a disease, disorder or condition arising from a topical microbialinfection, comprising administering to a subject in need thereof aneffective amount of (i) bicarbonate and (ii) the antimicrobial agent,wherein the antimicrobial agent's concentration in a cell of themicroorganism is increased by one or both of (1) decrease in pH gradientacross the microorganism's cytoplasmic membrane; and (2) increase in themicroorganism's cytoplasmic membrane potential. In some embodiments, theantimicrobial agent is a macrolide, tetracycline, peptide, glycopeptide,penicillin, cephalosporin, quinolone, fluoroquinolone, rifampin, innateimmunity factor, or combinations thereof. In some embodiments, theantimicrobial agent is not an aminoglycoside.

Also provided herein is a use of bicarbonate and an antimicrobial agentto treat a topical microbial infection, or a disease, disorder orcondition arising from a topical microbial infection, and a use ofbicarbonate and an antimicrobial agent to prepare a medicament to treata topical microbial infection, or a disease, disorder or conditionarising from a microbial infection, wherein the antimicrobial agent'sconcentration in a cell of the microorganism is increased by one or bothof (1) decrease in pH gradient across the microorganism's cytoplasmicmembrane; and (2) increase in the microorganism's cytoplasmic membranepotential. Also included is bicarbonate for use and an antimicrobialagent to treat a topical microbial infection, or a disease, disorder orcondition arising from a microbial infection, wherein the antimicrobialagent's concentration in a cell of the microorganism is increased by oneor both of (1) decrease in pH gradient across the microorganism'scytoplasmic membrane; and (2) increase in the microorganism'scytoplasmic membrane potential. In some embodiments, the antimicrobialagent is a macrolide, tetracycline, peptide, glycopeptide, penicillin,cephalosporin, quinolone, fluoroquinolone, rifampin, innate immunityfactor, or combinations thereof. In some embodiments, the antimicrobialagent is not an aminoglycoside.

Compositions, Pharmaceutical Uses and Routes of Administration

In some embodiments, provided herein is a composition comprising: aneffective amount of (i) bicarbonate and (ii) an antimicrobial agent. Insome aspects, a composition described herein further comprises apharmaceutically acceptable carrier, diluent or excipient.

In some embodiments, a composition disclosed herein comprises:

an effective amount of (i) bicarbonate and (ii) an antimicrobial agent;

wherein the antimicrobial agent is an antiviral agent, an antibioticagent, an anti-fungal agent, an anti-parasitic agent or an innateimmunity factor; and

wherein the antibiotic agent is a macrolide, a tetracycline, acephalosporin, a quinolone, a rifampin or a fluoroquinolone.

In some embodiments, a composition disclosed herein comprises:

an effective amount of (i) bicarbonate and (ii) an antimicrobial agent;

wherein the antimicrobial agent is an antiviral agent, an antibioticagent, an anti-fungal agent, an anti-parasitic agent or an innateimmunity factor; and

wherein the antibiotic agent is Amikacin, Neomycin, Tobramycin,Paromomycin, Streptomycin, Spectinomycin, Ertapenem, Doripenem,Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin, Cefalothin,Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime,Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime,Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftarolinefosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Clindamycin,Lincomycin, Lipopeptide, Daptomycin, Azithromycin, Clarithromycin,Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin,Telithromycin, Spiramycin, Aztreonam, Linezolid, Posizolid, Radezolid,Torezolid, Amoxicillin, Azlocillin, Carbenicillin, Cloxacillin,Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin,Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G,Temocillin, Ticarcillin, Bacitracin, Colistin, Polymyxin B,Besifloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide,Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine,Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine,Sulfisoxazole, Sulfonamidochrysoidine, Demeclocycline, Doxycycline,Minocycline, Oxytetracycline, Tetracycline, Arsphenamine,Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin,Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline,Tinidazole, Trimethoprim, Clofazimine, Dapsone, Capreomycin,Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide,Rifampicin, Rifabutin, Rifapentine or Streptomycin, or apharmaceutically acceptable salt thereof.

In some embodiments, the antimicrobial agent in a composition describedherein is erythromycin, dirithromycin, levofloxacin, norfloxacin,ciprofloxacin, enoxacin, moxifloxacin, besifloxacin or polymyxin B, or apharmaceutical salt thereof.

In some embodiments, the composition comprises sodium bicarbonate andindolicidin, bactenesin, alpha-defensin, bile salts, lysozyme,protegrin, or hyaluronic acid.

In some embodiments, the composition comprises about 1 mM to about 150mM bicarbonate.

In some embodiments, the antimicrobial agent is pentamidine, or apharmaceutically acceptable salt or analog thereof. In one embodiment, acomposition comprises an effective amount of (i) bicarbonate and (ii)pentamidine or a pharmaceutically acceptable salt thereof. In someembodiments, the composition comprises about 0.1 wt % to about 1.0 wt %pentamidine and about 0.01 wt % to about 1.0 wt % bicarbonate.

In some embodiments, the composition is suitable for administrationintraocularly. In some embodiments, the composition is useful as anophthalmic topical solution or gel, or is for topical, subconjunctival,periocular, retrobulbar, sub-tenon, intracameral, intravitreal,intraocular, subretinal, juxtascleral or suprachoroidal administration.

The bicarbonate and an antimicrobial agent can be administered to asubject, or used, in a variety of forms depending on the selected routeof administration, as will be understood by those skilled in the art.

In some embodiments, the bicarbonate and/or an antimicrobial agent canbe administered to the subject, or used, by oral (including sublingualand buccal) or parenteral (including, intravenous, intraperitoneal,subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary,intrathecal, rectal, topical, patch, pump, intraocular and transdermal)administration and the compound(s) formulated accordingly. In someembodiments, both the bicarbonate and the antimicrobial agent areadministered orally. In some embodiments, both the bicarbonate and theantimicrobial agent are administered parenterally. In some embodiments,the bicarbonate and the antimicrobial agent are administrated viadifferent modes of administration. In some embodiments, the bicarbonateis administered parenterally and the antimicrobial agent is administeredorally. In some embodiments, the bicarbonate is administered orally andthe antimicrobial agent is administered parenterally.

In one embodiment, the bicarbonate and an antimicrobial agent areadministered to the subject orally. In another embodiment, thebicarbonate and an antimicrobial agent are administered to the subjectintravenously.

Conventional procedures and ingredients for the selection andpreparation of suitable compositions are described, for example, inRemington's Pharmaceutical Sciences (2000-20th edition) and in TheUnited States Pharmacopeia: The National Formulary (USP 24 NF19)published in 1999. In general, the bicarbonate and an antimicrobialagent can be used in the forms in which they are available andadministered to subjects. Such forms, include, for example in the formof their pharmaceutically acceptable salts, in the form of fineparticles of the zwitterionic form and in an injectable or infusablesuspensions.

In some embodiments, the bicarbonate and/or the antimicrobial agent isadministered to a subject as an ophthalmic topical solution or gel, orby topical, dermal, transdermal, subconjunctival, periocular,retrobulbar, sub-tenon, intracameral, intravitreal, intraocular,subretinal, juxtascleral or suprachoroidal administration.

The pharmaceutical forms suitable for injectable use can include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In some embodiments, the form is sterile and fluid to theextent that easy syringability exists.

In some embodiments, parenteral administration can be by continuousinfusion over a selected period of time. Solutions suitable forparenteral administration can be prepared by known methods by a personskilled in the art. For example, the bicarbonate and/or an antimicrobialagent can be prepared in water optionally mixed with a surfactant suchas hydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, DMSO and mixtures thereof with or withoutalcohol, and in oils. Under ordinary conditions of storage and use,these preparations can contain a preservative to prevent the growth ofmicroorganisms.

Compositions for nasal administration can be conveniently formulated asaerosols, drops, gels or powders. Aerosol formulations typicallycomprise a solution or fine suspension of the active substance in aphysiologically acceptable aqueous or non-aqueous solvent and are can beprepared in single or multidose quantities in sterile form in a sealedcontainer, which take the form of a cartridge or refill for use with anatomising device. Alternatively, the sealed container can be a unitarydispensing device such as a single dose nasal inhaler or an aerosoldispenser fitted with a metering valve which is intended for disposalafter use. Where the dosage form comprises an aerosol dispenser, it cancontain a propellant which is, for example, a compressed gas such ascompressed air or an organic propellant such as fluorochlorohydrocarbon.In some embodiments, the aerosol dosage forms can take the form of apump-atomizer.

Compositions suitable for buccal or sublingual administration caninclude tablets, lozenges, and pastilles, wherein the active ingredientis formulated with a carrier such as sugar, acacia, tragacanth, gelatinand/or glycerine. Compositions for rectal administration canconveniently be in the form of suppositories containing a conventionalsuppository base such as cocoa butter.

In some embodiments provided herein, a composition useful for treatingan ophthalmic infection is provided. In some embodiments, thecomposition contains an effective amount (i) bicarbonate and (ii) anantimicrobial agent as provided herein and a pharmaceutical excipientsuitable for ocular administration. In some embodiments, pharmaceuticalcompositions suitable for ocular administration can be presented asdiscrete dosage forms, such as drops or sprays each containing apredetermined amount of the active ingredient(s) in a solution, or asuspension in an aqueous or non-aqueous liquid, an oil-in-wateremulsion, or a water-in-oil liquid emulsion. Other administration formscan include intraocular injection, intravitreal injection, topically, orthrough the use of a drug eluting device, microcapsule, implant, ormicrofluidic device. In some embodiments, the compounds as providedherein can be administered with a carrier or excipient that increasesthe intraocular penetrance of the compound such as an oil and wateremulsion with colloid particles having an oily core surrounded by aninterfacial film. The compositions of the invention can be administeredto the eye via topical, subconjunctival, periocular, retrobulbar,subtenon, intracameral, intravitreal, intraocular, subretinal,juxtascleral and suprachoroidal administration.

In some embodiments, eye drops can be prepared by dissolving the activeingredient(s) in a sterile aqueous solution such as physiologicalsaline, buffering solution, etc., or by combining powder compositions tobe dissolved before use. Other vehicles can be chosen, as is known inthe art, including, but not limited to: balance salt solution, salinesolution, water soluble polyethers such as polyethyene glycol,polyvinyls, such as polyvinyl alcohol and povidone, cellulosederivatives such as methylcellulose and hydroxypropyl methylcellulose,petroleum derivatives such as mineral oil and white petrolatum, animalfats such as lanolin, polymers of acrylic acid such ascarboxypolymethylene gel, vegetable fats such as peanut oil andpolysaccharides such as dextrans, and glycosaminoglycans such as sodiumhyaluronate. In some embodiments, additives ordinarily used in the eyedrops can be added. Such additives can include isotonizing agents (e.g.,sodium chloride, etc.), buffer agent (e.g., boric acid, sodiummonohydrogen phosphate, sodium dihydrogen phosphate, etc.),preservatives (e.g., benzalkonium chloride, benzethonium chloride,chlorobutanol, etc.), thickeners (e.g., saccharide such as lactose,mannitol, maltose, etc.; e.g., hyaluronic acid or its salt such assodium hyaluronate, potassium hyaluronate, etc.; e.g.,mucopolysaccharide such as chondroitin sulfate, etc.; e.g., sodiumpolyacrylate, carboxyvinyl polymer, crosslinked polyacrylate, polyvinylalcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propylmethylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxy propyl cellulose or other agents known to those skilled in theart).

In another embodiment, bicarbonate and/or an antimicrobial agent can beorally administered, for example, with an inert diluent or with anassimilable edible carrier, or they can be enclosed in hard or softshell gelatin capsules, or compressed into tablets, or incorporateddirectly with the food of a diet. For oral administration, thebicarbonate and an antimicrobial agent can be incorporated withexcipients and used in the form of, for example, ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Oral dosage forms also include modified release, forexample immediate release and timed-release, formulations. Examples ofmodified-release formulations include, for example, sustained-release(SR), extended-release (ER, XR, or XL), time-release or timed-release,controlled-release (CR), or continuous-release (CR or Contin), employed,for example, in the form of a coated tablet, an osmotic delivery device,a coated capsule, a microencapsulated microsphere, an agglomeratedparticle, e.g., molecular sieving type particles, or, a fine hollowpermeable fiber bundle, or chopped hollow permeable fibers, agglomeratedor held in a fibrous packet. In some embodiments, timed-releasecompositions can be, formulated, as liposomes or those wherein theactive compound is protected with differentially degradable coatings,such as by microencapsulation, multiple coatings, etc. Liposome deliverysystems include, for example, small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. In some embodiments,liposomes are formed from a variety of lipids, such as cholesterol,stearylamine or phosphatidylcholines.

It is also possible to freeze-dry the bicarbonate and/or anantimicrobial agent and use the lyophilizate obtained, for example, forthe preparation of products for injection

In some embodiments, the bicarbonate and/or an antimicrobial agent canbe coupled with soluble polymers as targetable drug carriers. Suchpolymers include, for example, polyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. In a further embodiment, thebicarbonate and antimicrobial agent can be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyglycolic acid, copolymers ofpolylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans,polycyanoacrylates and crosslinked or amphipathic block copolymers ofhydrogels.

In some embodiments, the bicarbonate and an antimicrobial agent areeffective for treating a pulmonary, lung, otic, oral, nasal, sinus,ophthalmic, intraocular, dermal, cardiovascular, kidney, urinary,gastrointestinal, rectal, vaginal or neurological infection, and thecompounds formulated accordingly. In some embodiments, the methods andcompositions described herein are useful to treat a pulmonary, lung,otic, oral, nasal, sinus, ophthalmic, intraocular, dermal,cardiovascular, kidney, urinary, gastrointestinal, rectal, vaginal orneurological infection.

In some embodiments, the bicarbonate and/or an antimicrobial agent canbe administered as a topical composition, such as a solution, gel,cream, lotion, liquid suspension, aerosol, nebulized spray, ointment,drops or patch.

Accordingly, in some embodiments, the bicarbonate and/or anantimicrobial agent can be administered intraocularly, for example, asan ophthalmic topical solution or gel (pulsed or sustained releaseddelivery), or by topical, subconjunctival, periocular, retrobulbar,sub-tenon, intracameral, intravitreal, intraocular, subretinal,juxtascleral or suprachoroidal administration.

In some embodiments, the bicarboniate and/or the antimicrobial agent arefound on a substrate. For example, a substrate may be glass, metal,plastic, latex or ceramic. In some aspects the bicarbonate and/or themicrobial agent may be present on or in a surgical instrument. In someembodiments, the bicarbonate and/or the antimicrobial agent may bepresent on or in a catheter, an implant, a stent or a surgical mesh.

The bicarbonate and an antimicrobial agent can be used with each other.The bicarbonate and an antimicrobial agent can be either used oradministered separately in time and/or in mode of administration (i.e.,different administration routes) or they can be administered together inthe same pharmaceutical preparation.

In some embodiments, bicarbonate and an antimicrobial agent can be usedor administered separately in time and/or in mode of administration. Forexample, the bicarbonate can be administered by injection and theantimicrobial agent can be administered orally. In another example, thebicarbonate can be administered orally and the antimicrobial agent canbe administered by injection. In a further example, both the bicarbonateand the antimicrobial agent can be administered by injection. When thebicarbonate and the antimicrobial agent are used or administeredseparately in time and/or in mode of administration, the bicarbonate canbe administered, or used, either before or after administration, or use,of the antimicrobial agent.

In some embodiments, bicarbonate and an antimicrobial agent can bepresent in same pharmaceutical composition or each can be present in aseparate composition. In one embodiment, bicarbonate and anantimicrobial agent are not present in the same composition.

The exact details of the administration will depend on thepharmacokinetics of the bicarbonate and the antimicrobial agent in thepresence of each other, and can include administering bicarbonate andthe antimicrobial agent within a few hours of each other, or evenadministering the bicarbonate and the antimicrobial agent within 24hours or greater of administration of the other, if the pharmacokineticsare suitable. Design of suitable dosing regimens is routine for oneskilled in the art.

In some embodiments, bicarbonate and an antimicrobial agent areadministered to a subject in a single composition or formulation. Insome embodiments, a single composition or formulation may comprisebicarbonate and one, two, three or more antimicrobial agents.

In some embodiments, bicarbonate and an antimicrobial agent are eachadministered to a subject in a separate composition or formulation.

Treatment methods can comprise administering to a subject, thebicarbonate and an antimicrobial agent, and optionally consists of asingle administration, or alternatively comprises a series ofadministrations. The length of the treatment period can depend on avariety of factors, such as the severity of the infection, disease,disorder or condition, the age of the subject, the dosage of thebicarbonate and an antimicrobial agent, the activity of the bicarbonateand an antimicrobial agent and/or a combination thereof.

In some embodiments, the antimicrobial agent can be administered or usedaccording to treatment protocol that is known for the antimicrobialagent in the treatment in microbial infections.

In some embodiments, the bicarbonate and an antimicrobial agent can beadministered or used as soon as practicable after exposure to themicroorganism. In some embodiments, the bicarbonate and an antimicrobialagent can be administered or used until treatment of the microbialinfection is achieved. For example, until complete elimination of themicroorganism is achieved, or until the number of microorganisms hasbeen reduced to the point where the subject's defenses are no longeroverwhelmed and can kill any remaining microorganisms.

The dosage of the bicarbonate and an antimicrobial agent can varydepending on many factors such as the pharmacodynamic propertiesthereof, the mode of administration, the age, health and weight of thesubject, the nature and extent of the symptoms, the frequency of thetreatment and the type of concurrent treatment, if any, and theclearance rate in the subject to be treated. One of skill in the art candetermine the appropriate dosage based on the above factors. In someembodiments, the bicarbonate and an antimicrobial agent are administeredinitially in a suitable dosage that may be adjusted as required,depending on the clinical response.

In some embodiments, the dosage, or effective amount, of theantimicrobial agent can be equal to or less than the dosage of suchagents when used alone. Such dosages are known to or readily determinedby those skilled in the art.

In some embodiments, the dosage or amount of the bicarbonate can be anamount to provide physiological concentrations of bicarbonate, or about25 mM of bicarbonate In some embodiments, the bicarbonate can beadministered in a composition comprising about 1 mM to about 75 mMbicarbonate, or about 20 mM to about 50 mM bicarbonate. In someembodiments, the bicarbonate can be administered in a compositioncomprising about 0.01 wt % to about 1.0 wt % bicarbonate, or about 0.20wt % to about 0.5 wt % bicarbonate. In some embodiments, the effectiveamount or dosage of bicarbonate can be about 0.01 mg to about 1 mg.

In some embodiments, provided herein is a pharmaceutical compositioncomprising sodium bicarbonate and an antimicrobial agent wherein theantimicrobial agent's concentration in a cell of the microorganism isincreased by one or both of (1) decrease in pH gradient across themicroorganism's cytoplasmic membrane; and (2) increase in themicroorganism's cytoplasmic membrane potential. In some embodiments, thecomposition comprises about 1 mM to about 150 mM bicarbonate, or about20 mM to about 50 mM bicarbonate.

In illustrative embodiments, the antimicrobial agent is pentamidine,erythromycin, dirithromycin, levofloxacin, norfloxacin, ciprofloxacin,enoxacin, moxifloxacin, besifloxacin or polymyxin B, combinationthereof, analogs thereof, or a pharmaceutically acceptable salt of, andthe composition comprises about 0.1 wt % to about 1.0 wt %, or about 0.5wt %, pentamidine and about 0.01 wt % to about 1.0 wt % bicarbonate, orabout 0.20 wt % to about 0.5 wt % bicarbonate.

Screening for Antimicrobial Agents

Provided herein are methods of screening for antimicrobial agents orcompounds.

In one embodiment, the method of screening for an antimicrobial agentcomprises (1) contacting a microorganism with a test compound in thepresence of bicarbonate; and (2) observing growth of the microorganism,wherein a decrease in the growth of the microorganism in the presence ofthe test compound compared to in the absence of the test compoundindicates that the test compound is an antimicrobial agent.

In another embodiment, provided herein are methods of screening forantimicrobial compounds that can be modulated by bicarbonate comprising:(1) contacting a microorganism with either (i) a test compound not inthe presence of bicarbonate or (ii) a test compound in the presence ofbicarbonate; and (2) observing growth of the microorganism, wherein agreater change in the growth of the microorganism with the test compoundin the presence of bicarbonate compared to the growth of themicroorganism with the test compound not in the presence of bicarbonateindicates that the test compound is an antimicrobial compound that canbe modulated by bicarbonate.

In some embodiments, a microorganism is a virus, a bacterium, a fungusor a parasite.

In some embodiments, the concentration of bicarbonate is about 20 mM toabout 75 mM, or about 25 mM to about 50 mM, or about 20 mM to about 100mM, or about 20 mM to about 150 mM, or about 50 to about 150 mM, orabout 50 mM to about 100 mM.

In some embodiments a plurality of test compounds may be tested. In someembodiments, a plurality of microorganisms may be tested.

In some embodiments, the method of screening can be performed atdifferent concentrations of the test compound and a minimumconcentration for inhibition (MIC) of the microorganism is determined.In some embodiments, the MIC is the lowest concentration of a compoundthat prevents visible growth of the microorganism after overnightincubation.

In some embodiments, a FIC index calculation (shown below) is used toscreen for an antimicrobial agent. In some embodiments, a FIC indexvalue of less than or equal to 0.5 indicates synergy between a testcompound and bicarbonate.FIC index=FIC_(bicarbonate salt)+FIC_(test compound)Fractional Inhibitory Concentration (FIC)=[X]/MIC_(X), where [X] is thelowest inhibitory concentration of drug in the presence of the co-drug.

EXAMPLES

The following examples illustrate the scope of the application. Specificelements of the example are for descriptive purposes only and are notintended to limit the scope of the invention. Those skilled in the artcould develop equivalent methods and utilize comparable materials thatare within the scope of the application.

Example 1: Methods

In the following experiments, bacterial cells were cultured in 96-wellmicrotiter plates in cation-adjusted Mueller-Hinton Broth (MHB) for 18 hat 37° C. The main strains used in this study were E. coli (K-12BW25113) (placed in stationary incubator) and S. aureus (Strain Newman)(incubated at 250 r.p.m). Knockout strains (ΔtolcC and ΔychM) were usedfrom the Keio knockout collection (Baba, T. et al. Construction ofEscherichia coli K-12 in-frame, single-gene knockout mutants: the Keiocollection. Mol Syst Biol 2, 2006 0008, doi:10.1038/msb4100050 (2006)).For minimum concentration for inhibition (MIC) determination andcheckerboard analyses, Clinical & Laboratory Standards Institute (CLSI)protocol was used. Tetracycline uptake was assayed as previouslydescribed (Ejim, L. et al. Combinations of antibiotics and nonantibioticdrugs enhance antimicrobial efficacy. Nature chemical biology 7,348-350, doi:10.1038/nchembio.559 (2011)). DiSC₃ loading of S. aureuscells was performed as previously described (Farha, M. A., Verschoor, C.P., Bowdish, D. & Brown, E. D. Collapsing the proton motive force toidentify synergistic combinations against Staphylococcus aureus.Chemistry & biology 20, 1168-1178, doi:10.1016/j.chembiol.2013.07.006(2013)). CCCP concentration of 20 μM was used. pH adjustments were madeby addition of HCl or NaOH. INT assay and ATP bioluminescence assayswere performed as previously described (Farha, M. A., Verschoor, C. P.,Bowdish, D. & Brown, E. D. Collapsing the proton motive force toidentify synergistic combinations against Staphylococcus aureus.Chemistry & biology 20, 1168-1178, doi:10.1016/j.chembiol.2013.07.006(2013)). For chemical-genomic studies, the Keio library was grownovernight in cation-adjusted MHB broth, in 384-well plates. From these,treatment plates (25 mM sodium bicarbonate, or a sterile water control)were inoculated and grown for 15 hours at 37° C., in a stationaryincubator. Data was normalized according to Mangat et al (Mangat, C. S.,Bharat, A., Gehrke, S. S. & Brown, E. D. Rank ordering plate datafacilitates data visualization and normalization in high-throughputscreening. J Biomol Screen 19, 1314-1320, doi:10.1177/1087057114534298(2014)), with gene products and GO terms mined from EcoCyc. The GFPpromoter library (Keseler, I. M. et al. EcoCyc: fusing model organismdatabases with systems biology. Nucleic Acids Res 41, D605-612,doi:10.1093/nar/gks1027 (2013)) was grown for 18 hours at ambienttemperature with the same inoculation strategy as with the Keiocollection. The analysis pipeline of Zaslaver et al (Zaslaver, A. et al.A comprehensive library of fluorescent transcriptional reporters forEscherichia coli. Nat Methods 3, 623-628, doi:10.1038/nmeth895 (2006))was used to generate maps of promoter activity, from which lists ofpromoters with increased or decreased activity were compiled.

Example 2: Pentamidine Possesses Monotherapy Activity in a Murine Modelof Systemic A. baumannii Infection

Pentamidine (Formula I) has been recognized as having antibacterialactivity for approximately 70 years but has not yet been pursued as anantibacterial in the clinic. Knowledge that the concentrations ofpentamidine required for inhibiting the growth of bacteria in vitro arehigh.

A screen of a collection of previously-approved non-antibiotic drugs toidentify small molecule perturbants of outer membrane architectureuncovered the antiprotozoal/antifungal drug pentamidine (I). The screenwas devised based on observations that E. coli grown during periods ofcold stress is rendered susceptible to glycopeptide antibiotics, aphenomenon that can be reversed by inactivating genes involved in outermembrane biosynthesis (Stokes, J M., et al., Cell Chem. Biol., Feb. 18,2016, 23(2), 267-277).

Using atomic force microscopy (AFM), the direct effect of pentamidine onthe outer membrane of E. coli has been observed for the first time.Severe surface defects were observed upon incubation with pentamidinethat are similar to those seen with the membrane-damaging agent,polymyxin B. Structure-activity relationship studies have providedfurther support for these observations and suggested that the cationicproperties of pentamidine may promote its interaction with the anionichead groups of the bacterial membrane lipids, and enable integrationinto the hydrophobic core of the membrane. This indicated that membraneperturbation is the principal mechanism of action of pentamidine.

Here, A. baumannii was injected into mice intraperitoneally (1.5×10⁶ CFUA. baumannii) and after two-hours mice were treated intraperitoneallywith a single dose of PBS (n=10), novobiocin at 5 mg/kg (n=10),pentamidine isethionate at 10 mg/kg (n=10), pentamidine at 50 mg/kg(n=10) or a combination of both molecules where pentamidine is at 10mg/kg and novobiocin, 5 mg/kg (n=10). Mice were treated two-hourspost-infection to allow complete organ occupancy by the injected cells.

FIG. 1A shows that when mice were treated IP with 50 mg/kg pentamidinealone, rescue in 80% of the animals was observed. FIG. 1B shows that A.baumannii organ load (e.g. spleen) was found to decrease seven logs as aresult of pentamidine alone, and total clearance as a result of thecombination.

Example 3: Pentamidine Activity in Different Culture Media

While pentamidine had relatively little or no in vitro activity instandard microbiological media against the Gram-negative organisms, E.coli, A. baumannii, K pneumoniae, P. aeruginosa and B. cenocepacia,these bacteria were highly susceptible to pentamidine when grown in atissue culture media formulated to mimic the natural environment (Table1). For instance, the minimum inhibitory concentration (MIC) ofpentamidine against E. coli in the standard microbiological mediaMueller Hinton Broth (MHB) of 200 μg/mL was significantly reduced in thetissue culture media formulated to mimic the natural environment to 1μg/mL. Against A. baumannii, a >50-fold enhanced potency in tissueculture media formulated to mimic the natural environment was observed.Potent activity was also observed against Gram-positive organisms, suchas S. aureus.

TABLE 1 Effects of pentamidine Tissue culture media MHB formulated tomimic the natural (μg/mL) environment (μg/mL) Escherichia coli 200 1.5Acinetobacter baumannii 100 12.5 Klebsiella pneumoniae >200 12.5Pseudomonas aeruginosa >200 12.5 Burkhloderia cenocepacia >200 12.5Burkhloderia multivorans >200 12.5 Enterococcus faecalis 25 3.1Staphylococcus aureus 12.5 <0.1

Example 4: Sodium Bicarbonate Potentiated the Activity of Pentamidine,in a Dose-Dependent Manner, Against E. coli

The components of the tissue culture media formulated to mimic thenatural environment were deconvoluted and tested at varyingconcentrations to assess their individual effects on the antibacterialactivity of pentamidine. Most notably, sodium bicarbonate, which ispresent at physiological concentrations (25 mM) in the tissue culturemedia formulated to mimic the natural environment, and absent instandard microbiological media, greatly potentiated the activity ofpentamidine, in a dose-dependent manner, against E. coli (FIG. 2 ). Theability of bicarbonate to potentiate the activity of pentamidine largelyreconciled the paradox between the lack of activity observed in standardmedia in vitro and the significant in vivo activity of pentamidine as asingle agent. Importantly, bicarbonate plays a central position inmammalian physiology and is ubiquitously present in the body.

Table 2 shows the effect of various salts on the antibacterial activityof pentamidine against S. aureus. Shown are the Fractional InhibitoryConcentration (FIC) indexes from the combination of pentamidine witheach salt where FICI≤0.5 represents synergy, =1-2 additivity and >4antagonism. Through the testing of various salts, with differing ionicstrengths and other differing properties, on the activity ofpentamidine, it was observed that sodium bicarbonate, was unique andprovided the greatest influence on the activity of pentamidine. Thesodium counterion did not contribute to the potentiation of pentamidine,as equally potent synergy was observed with varying salts ofbicarbonate.

TABLE 2 Effects of various salts on the antibacterial activity ofpentamidine against S. aureus FIC Index (with pentamidine in S. aureus)Sodium bicarbonate (NaHCO₃) 0.31 Ammonium bicarbonate (NH₄HCO₃) 0.28Sodium bromide (NaBr) 2 Sodium fluoride (NaF) 1 Sodium acetate(C₂H₃NaO₂) 2 Sodium sulfate (Na₂SO₄) 2 Sodium chloride (NaCl) 2Isethionic acid (C₂H₆O₄S) 2 Boric acid (H₃BO₃) 1 Sodium nitrate (NaNO₃)2 Sodium phosphate (NaH₂PO₄) >8 Potassium phosphate (KH₂PO₄) >8 FICindex = FIC_(salt) + FIC_(pentamidine) Fractional InhibitoryConcentration (FIC) = [X]/MIC_(X), where [X] is the lowest inhibitoryconcentration of drug in the presence of the co-drug.

Example 5: The Impact of Pentamidine on the Proton Motive Force of E.coli MC1061

Fluorescence spectroscopy using the membrane-potential sensitive dye3,3′-dipropylthiadicarbocyanine iodide was used to measure pentamidine'sability to dissipate transmembrane potential. E. coli MC1061 cells werewashed twice and suspended in a buffer containing 20 mM glucose and 5 mMHEPES (pH 7.2). Final resuspension was diluted to an optical density at600 nm of 0.085. DiSC₃(5) was added at a final concentration of 1 andthe dye was left to stabilize (1 hr at 37° C.). Compounds were theninjected at concentrations equivalent to their MIC. Fluorescent traceswere measured in a fluorimeter (Photon Technology International) at theexcitation and emission wavelengths of 622 and 660 nm, respectively.

It was reasoned that the much-reduced MIC of pentamidine in the presenceof an ionic milieu reminiscent of the in vivo environment might indicatean alternate mode of action than observed in standard media. Recognizingthat the ability of pentamidine to perturb the outer membrane wasretained in standard microbiological media, it was queried whether theionic environment of the host might confer on pentamidine additionalactivity against the cytoplasmic membrane. To test this, the impact ofpentamidine on the proton motive force (PMF) of E. coli wasinvestigated. Fluorescence spectroscopy using the membrane-potentialsensitive dye 3,3′-dipropylthiadicarbocyanine iodide revealed thatpentamidine dissipates transmembrane potential. Briefly, due to thepotential gradient, the dye is taken up by bacteria and accumulation inthe membrane leads to a decrease in fluorescence intensity due toself-quenching. A subsequent increase in fluorescence intensity isobserved only if the dye is displaced into the solution as a result ofdissipation of the membrane potential. Pentamidine dissipated themembrane potential of E. coli whether grown in standard growth media orwhen supplemented with 25 mM sodium bicarbonate (FIG. 3A, FIG. 3B).While not wishing to be limited by theory, the observation thatpentamidine is synergistic with sodium bicarbonate may be related to itsability to dissipate the psi component of PMF (Δψ), whereas sodiumbicarbonate can dissipate the pH gradient leading to a complete collapseof PMF.

Example 6: Pentamidine is Effective for Inhibiting the Growth of BothGram-Negative and Gram-Positive Pathogens

The potential of pentamidine as a therapeutic agent was tested in avariety of superficial skin infection models. In this model, aninfection is established by disrupting the skin barrier through partialremoval of the epidermal layer by stripping with adhesive tape and withsubsequent application of the pathogen. Tape-stripped mice infected with4×10⁶ CFU/mL A. baumannii (FIG. 4A) or 4×106 CFU/mLmethicillin-resistance Staphylococcus aureus (MRSA) USA-300 (FIG. 4B).The No Infection group (n=1) shows the natural bacterial load aftertape-stripping with no inoculum applied. Twenty μL of 0.5% Pentamidinein 1.9% Boric Acid, pH 7.0 was applied to the wound area 4,5,6,7,8,9,and 19,20,21,22,23,24 hours post infection (n=4). This treatment regimewas also conducted for the Vehicle (1.9% Boric Acid, pH 7) as a control(n=4). Tissue samples were collected at 25 hr post-infection (FIG. 4A)and 28 hr post-infection (FIG. 4B). In the A. baumannii skin infectionmodel, where mice were treated with 0.5% pentamidine, a 4-log reductionin CFU/mL compared to vehicle-treated mice was observed (FIG. 4A). In aS. aureus skin infection model, 0.5% pentamidine treatment caused a5-log reduction in CFU/mL (FIG. 4B). Further, addition of bicarbonate inthe solution increased the antibacterial efficacy of pentamidine inclearing an MRSA infection (pentamidine was applied as a topical 0.5%aqueous solution on skin with or without bicarbonate (50 mM)). (FIG. 5). Overall, pentamidine offers an effective, localized andwell-tolerated topical approach for both Gram-negative and Gram-positivepathogens.

Example 7: The Effect of Addition of Sodium Bicarbonate on ConventionalAntibiotics

The minimum inhibitory concentrations (MIC) of the antibiotics listed inFIG. 6A were determined in Mueller-Hinton broth (MHB) media and comparedto the MIC in MHB supplemented with physiological concentrations ofsodium bicarbonate (25 mM) (MHB+25 mM Sodium bicarbonate). Results ofdifferent experiments are shown in FIGS. 6A and 6B. Fold enhancement inMICs in the media supplemented with sodium bicarbonate is represented bya positive fold, whereas suppression of activity is represented by anegative fold.

FIG. 6A shows the fold changes in Escherichia coli in one experiment.The trends were similar in the Gram-positive Staphylococcus aureus.Additional data are shown in FIG. 6B and Table 3: the fold enhancementin the minimum inhibitory concentration (MIC) for a variety ofantibiotics in standard microbiological media relative to mediasupplemented with 25 mM sodium bicarbonate is shown for E. coli and S.aureus. As shown in FIG. 6B, eight classes of antibiotics investigatedhad appreciably altered activities in the presence of 25 mM sodiumbicarbonate. In FIG. 6B, the minimum inhibitory concentrations (MIC) ofthe listed antibiotics were determined in Mueller-Hinton broth (MHB)media and compared to the MIC in MHB supplemented with physiologicalconcentrations of sodium bicarbonate (25 mM). Fold enhancement in MICsin the media supplemented with sodium bicarbonate is represented by apositive value, whereas suppression of activity is represented by anegative value. Shown are the fold changes in E. coli (solid) and foldchanges in S. aureus (checkered).

With a few exceptions, these Gram-negative and Gram-positive bacteriabehaved similarly. Of the antibiotics tested, the antibacterial activityof some fluoroquinolones, macrolides, and aminoglycosides was enhanced.The activity of polymyxin B was enhanced strictly in S. aureus. Incontrast, the antibacterial activity of other fluoroquinolones, variouscell wall active drugs, tetracyclines, fosfomycin and novobiocin wassuppressed in the presence of bicarbonate. The antibacterial effect onother classes such as chloramphenicol, linezolid, the antifolate drugs,trimethoprim and sulfamethoxazole, remained largely unchanged.

TABLE 3 MIC of various antibiotics in MHB vs MHB + 25 mM sodiumbicarbonate against E. coli and S. aureus. E. coli S. aureus MHB− MHB+Fold MHB− MHB+ Fold Apramycin 32 8 4 16 32 0.5 Gentamicin 2 0.25 8 2 1 2Kanamycin 4 1 4 8 4 2 Neomycin 1 0.25 4 2 0.5 4 Paromomycin 2 1 2 2 2 1Spectinomycin 16 4 4 64 8 8 Chloramphenicol 8 8 1 8 8 1 Dirithromycin128 4 32 4 0.5 8 Erythromycin 128 32 4 32 1 32 Doxycycline 1 4 −4 0.1252 −16 Tetracycline 1 4 −4 0.5 2 −4 Linezolid 256 256 1 0.625 0.625 1Bacitracin >256 >256 1 32 64 −2 Fosfomycin 4 16 −4 16 64 −4 Fosmidomycin16 32 −2 >64 >64 1 Ampicillin 16 32 −2 1 8 −8 Amoxicillin 8 16 −2 0.25 1−4 Cloxacillin 256 256 1 0.031 0.5 −16 Piperacillin 2 1 2 0.25 1 −4Oxacillin 256 >256 −2 0.0625 1 −16 Ceftriaxone 0.625 0.625 1 4 16 −4Cefoperazone 0.125 0.125 1 0.25 4 −16 Vancomycin 256 256 1 1 4 −4Polymyxin B 0.25 0.5 −2 256 32 8 Ciprofloxacin 0.0625 0.0156 4 0.5 1 −2Besifloxacin 0.25 0.0313 8 0.125 0.25 −2 Enoxacin 0.25 0.125 2 1 2 −2Nalidixic acid 2 8 −4 >8 32 −4 Norfloxacin 0.125 0.0625 2 0.5 1 −2Levofloxacin 0.0313 0.0313 1 0.5 1 −2 Moxifloxacin 0.0313 0.0156 2 0.1250.0625 2 Pefloxacin 0.125 0.5 −4 0.5 2 −4 Novobiocin 32 >256 −8 0.0310.5 −16 Rifampicin 32 64 −2 0.0078 0.031 −4

Where antibiotics were potentiated or suppressed, follow-up studiesusing systematic microbroth checkerboard techniques were completed toassess the dose-dependence of the interaction (FIG. 6C). Indeed, in allcases, enhancement or suppression was further pronounced with increasingconcentrations of sodium bicarbonate. FIG. 6C shows representativeantibiotics whose activity was altered in the presence of 25 mM sodiumbicarbonate. The extent of inhibition is shown as a heat plot, such thatthe darkest color represents full bacterial growth.

Example 8: Sodium Bicarbonate Diminishes the Uptake of Tetracycline inE. coli

The concentration of tetracycline was 125 μg/ml, and concentration ofsodium bicarbonate was as indicated in FIG. 7 . Tetracycline uptake wasassayed by monitoring the fluorescence enhancement of tetracycline whenit enters the cell. Averages of duplicate experiments are shown in FIG.7 . In a similar experiment, a direct test of the cellular uptake oftetracycline revealed that that suppression observed was due toinhibition of tetracycline uptake on addition of bicarbonate (FIG. 11 ,Panel a). In Panel a, sodium bicarbonate diminished the uptake oftetracycline in E. coli. Concentration of tetracycline was 125 μg/ml,and concentration of sodium bicarbonate was as indicated. Tetracyclineuptake was assayed by monitoring the fluorescence enhancement oftetracycline when it enters the cell. Averages of triplicate experimentsare shown.

Example 9: Bicarbonate Potentiates the Action of Dirithromycin

Results shown in FIG. 8A show that a growth inhibitory concentration ofthe macrolide dirithromycin is potentiated in the presence of sodiumbicarbonate in wild-type E. coli and in FIG. 8B, show potentiation bysodium bicarbonate disappears in a strain deficient for the main effluxpumps E. coli ΔtolC.

The enhancement of dirithromycin by sodium bicarbonate was furtherassessed for the pathogens Acinetobacter baumannii, Klebsiellapneumoniae and Pseudomonas aeruginosa (FIG. 8C, Panels a-c). FIG. 8C,Panels a-c show the combination of the macrolide, dirithromycin, andsodium bicarbonate against multi-drug resistant clinical isolates of (a)Acinetobacter baumannii (b) Klebsiella pneumoniae and (c) Pseudomonasaeruginosa. In all cases, bicarbonate was a potentiator of the action ofthis macrolide antibiotic.

Example 10: Bicarbonate is Responsible for the Enhancements Observed

Whether the chemical bicarbonate was responsible for the enhancementsobserved in the above examples was assessed. It was observed that theactivity was not due to simply an effect on pH. Test media werepH-adjusted upon addition of sodium bicarbonate for all studies reportedherein. Of note, sodium bicarbonate at physiological concentration (25mM) produced media with a pH typical of standard susceptibility testingconditions (Table 4).

TABLE 4 pH of MHB media amended with various concentrations of sodiumbicarbonate, prior to pH-adjusting to 7.4. Concentration (mM) pH 0 7.421.56 7.42 3.12 7.42 6.25 7.43 12.5 7.47 25 7.53 50 7.61 100 7.71

Further, using dirithromycin, many equimolar organic salts were tested,with differing ionic strengths and steric properties, and none hadimpact on antibacterial activity, ruling out osmotic-mediated mechanisms(Table 5). Lastly, sodium did not contribute to the potentiation ofdirithromycin, as equally potent synergy was observed with other saltsof bicarbonate (Table 5).

TABLE 5 Minimum inhibitory concentration of dirithromycin in thepresence of various salts at 25 mM against E. coli. In all cases, pH wasadjusted to 7.4. Salt Formula MIC (μg/mL) Control — 128 Sodiumbicarbonate NaHCO₃ 4 Sodium bromide NaBr 128 Sodium chloride NaCl 128Sodium fluoride NaF 128 Sodium nitrate NaNO₃ 128 Sodium acetate C₂H₃NaO₂64 Sodium sulfate Na₂SO₄ 128 Isethionic acid C₂H₆O₄S 128 Boric acidH₃BO₃ 128 Sodium phosphate NaH₂PO₄ >128 Potassium phosphate KH₂PO₄ >128Ammonium bicarbonate KHCO₃ 8 Potassium bicarbonate NH₄HCO₃ 4

Example 11: Minimum Inhibitory Concentration of Pentamidine AgainstSaccharomyces cerevisiae

An overnight culture of Saccharomyces cerevisiae was diluted 1:5,000into fresh YPD medium, supplemented with 0, 25 mM or 50 mM sodiumbicarbonate and tested against two-fold serial dilutions of pentamidine.Plates were incubated 24 hours and optical density read at 600 nm

FIG. 9 shows that the MIC of pentamidine is reduced from 256 μg/mL to 4μg/mL in the presence of 25 mM sodium bicarbonate and to 2 μg/mL in thepresence of 50 mM sodium bicarbonate.

Example 12: Physiological Concentrations of Bicarbonate Enhance theAntibacterial Activity of Various Chemical Factors Involved in InnateImmunity

The influence of sodium bicarbonate (pH 7.4) on the in vitroantibacterial activity of various secretory molecules and cellularcomponents that make up innate immunity against bacterial pathogens wasinvestigated. Specifically, the ability of sodium bicarbonate, at thesub-MIC but physiological concentration of 25 mM, to potentiate theactivity of various mediators of host defense, including defensins andcathelicidins, whose family members make up the principal components ofinnate immunity in vertebrates (Zasloff, M. N Engl J Med 2002, 347:1199-1200), was assessed.

Sodium bicarbonate itself exhibited antibacterial activity against E.coli, Staphylococcus aureus, and other clinically relevant pathogens,with minimum inhibitory concentration (MIC) values ranging from 50-100mM (Table 6).

TABLE 6 MIC of sodium bicarbonate against various pathogens. OrganismMIC (mM) Escherichia coli 100 Staphylococcus aureus 50-100 Klebsiellapneumoniae 100 Acinetobacter baumannii  50 Pseudomonas aeruginosa >100 Enterococcus faecium 50-100

Shown in FIG. 10 , Panels a-e are potency analyses of various componentsagainst E. coli in MHB (line with Xs) and MHB supplemented with 25 mMsodium bicarbonate (line with circles) for a, LL-37; b, indolicidin; c,bactenesin; d, alpha-defensin; e, bile salts; f, lysozyme; g, protegrin;and h, hyaluronic acid. Averages of triplicate experiments are shown.

The antimicrobial activity of alpha-defensin and LL-37 were enhanced onaverage 4 to 8-fold against E. coli (FIG. 10 , Panels a,e) and S. aureus(Table 7). Other antimicrobial peptides, such as indolicidin andbactenesin, were also potentiated in the presence of bicarbonate, 128-and 256-fold, respectively against E. coli (FIG. 10 , Panels b,c), and16- and 256-fold, respectively against S. aureus (Table 7). Alsoenhanced in the presence of bicarbonate, was the activity of theporcrine leukocyte protegrin (8-fold in both E. coli and S. aureus)(FIG. 10 , Panel d; Table 7). Additionally, a physiologicalconcentration of sodium bicarbonate enhanced the inhibitory activity ofother innate immunity chemical factors such as lysozyme and bile saltsagainst E. coli (FIG. 1 , Panels f,g). The innate immunity chemicalbarrier, hyaluronic acid, which is ubiquitously expressed in theextracellular matrix of all vertebrate tissues was also potentiated inthe presence of sodium bicarbonate, 64-fold in both E. coli and S.aureus (FIG. 10 , Panel h; Table 7). It is noted that common among thesecomponents of innate immunity is their ultimate action on thecytoplasmic membrane causing membrane depolarization.

TABLE 7 MIC of various components of innate immunity in MHB vs MHB + 25mM sodium bicarbonate against S. aureus (strain Newman) MIC (μg/mL) MIC(μg/mL) in MHB + 25 Component in MHB mM bicarbonate LL-37 128 32 Indolicidin 128 8 Bactenesin 128   0.5 α-defensin ND* ND* Protegrin  324 Lysozyme  >256** >256**  Bile salts 512 512  Hyaluronic acid  4   0.0625 *ND: not determined; **S. aureus is intrinsically resistant tolysozyme

Example 13: The Effects of Proton Motive Force (PMF) Perturbations onthe Activity of Antibacterial Agents

The proton motive force (PMF) describes the electrochemical potential atthe cytoplasmic membrane that is composed of an electrical potential(Δψ, negative inside) and a proton gradient (ΔpH, acidic outside). It isknown that tetracyclines penetrate bacterial cells in a ΔpH-dependentmanner, while positively charged aminoglycosides utilize the Δψcomponent for transport. Agents that selectively perturb either Δψ orΔpH are known to prompt a compensatory increase in the other componentin order to maintain PMF. The role of bicarbonate in perturbing PMF ofbacteria was further assessed.

Treatment of E. coli with 25 mM bicarbonate led to a highertransmembrane distribution of 3,3′-dipropylthiadicarbocyanine iodide(DiSC₃(5)), a fluorescent probe that exhibits Δψ-dependent membraneaccumulation (FIG. 12 ). Without being bound to theory, this isconsistent that with the observed increase in aminoglycoside activity,selective dissipation of ΔpH by sodium bicarbonate is compensated for byan increase in Δψ that in turn drives uptake of aminoglycosides.

Specifically, FIG. 12 shows uptake of 3,3′-Dipropylthiacarbocyanineiodide, a membrane-potential sensitive dye. S. aureus cells were grownto exponential phase in the absence (untreated) or presence of 25 mMsodium bicarbonate (treated), washed and loaded with 1 μM DiSC₃(5). S.aureus treated with 25 mM sodium bicarbonate exhibited an increaseduptake in the levels of DiSC₃(5) as measured by fluorescence (blackcircles) and compared to uptake levels of untreated cells (whitecircles). Uptake and fluorescence was stable over time as shown in thegraph.

Pre-incubation of E. coli with the proton ionophore, carbonyl cyanidem-chlorophenyl hydrazone (CCCP; selectively targets the pH gradient ofcells), prior to treatment with sodium bicarbonate and gentamicin,reversed the potentiation observed (FIG. 11 , Panel b). Panel b showsthat pre-treatment with CCCP abolishes the potentiation of gentamicin bysodium bicarbonate. Shown are potency analyses of gentamicin in MHBagainst E. coli (1); MHB supplemented with 25 mM sodium bicarbonate (2);CCCP pre-treated cells in MHB (3); CCCP pre-treated cells in MHBsupplemented with 25 mM sodium bicarbonate (4). Averages of triplicateexperiments are shown.

Taken together, these data show that sodium bicarbonate influences theentry of antibacterial agents that are driven by PMF, suppressing thosethat require an intact pH gradient across the cytoplasmic membrane, andenhancing those that are driven by Δψ, such as the polycationicaminoglycosides.

Example 14: Effects of Bicarbonate on Cellular Respiration of Bacterialin the Context of Antibiotics

Other antibiotics that rely on cellular energetics for entry includefosfomycin and novobiocin. Fosfomycin is actively transported via aglycerol-3-phosphate permease where transport activity has been shown tobe dependent on ΔpH. Uptake of novobiocin is similarly an activetransport mechanism supported by ΔpH such that uncouplers and inhibitorsof respiration have been shown to reduce its cellular accumulation.Sodium bicarbonate suppressed the activity of fosfomycin and novobiocin(FIG. 6C).

Nigericin, an ionophore that selectively dissipates the pH gradient,also suppressed the activity of fosfomycin and novobiocin (FIG. 13 ,Panels a-b). Specifically, FIG. 13 , Panel a and FIG. 13 , Panel b showthat a combination of nigericin, a protonophore, with fosfomycin (Panela) or novobiocin (Panel b) leads to antagonistic interactions against S.aureus (sensitive to nigericin). Shown are microdilution checkerboardanalyses, where the extent of inhibition is shown as a heat plot, suchthat the darkest color represents full bacterial growth.

Fluoroquinolone (FQ) antibiotics show a variety of responses in thepresence of 25 mM sodium bicarbonate depending on their physicochemicalproperties and the organism in question. While convention holds that FQuptake is a passive process, previous studies have noted that theaddition of the protonophore CCCP results in increased uptake of someFQs, suggesting a role for the Δψ component of the proton motive force(Piddock, L. J., Jin, Y. F., Ricci, V. & Asuquo, A. E. Quinoloneaccumulation by Pseudomonas aeruginosa, Staphylococcus aureus andEscherichia coli. J Antimicrob Chemother 43, 61-70 (1999); and Diver, J.M., Piddock, L. J. & Wise, R. The accumulation of five quinoloneantibacterial agents by Escherichia coli. J Antimicrob Chemother 25,319-333 (1990)). Consistent with this, a potentiation of various FQs bybicarbonate was observed. The activity of FQs in the presence ofbicarbonate against E. coli correlated with the nature of thesubstituents at the C-7 position of the quinolone nucleus (Table 8). Theactivities of FQs containing more basic substituents at C-7 (e.g.ciprofloxacin and besifloxacin) increased in the presence ofbicarbonate, while those with more acidic substituents (e.g. nalidixicacid and pefloxacin) were suppressed (FIG. 6B, Table 8). These resultsindicate that the electrochemical component (Δψ) of the proton motiveforce has a role in FQ uptake. Compensatory increases in Δψ associatedwith dissipation of ΔpH by bicarbonate would favor the uptake ofpositively charged species. In S. aureus, however, there was noenhancement by bicarbonate of FQs; instead, a small suppression wasobserved for the antibacterial activity of this chemical class.

TABLE 8 Structural formula and physicochemical properties offluoroquinolones. Listed are the pKa values for the acidic and basicfunctions of the fluoroquinolones, generated from ChemAxon, aphysico-chemical property predictor. pKa (Strongest pKa (StrongestStructure Acidic) Basic) Besifloxacin

5.64 9.67 Ciprofloxacin

5.76 8.68 Enoxacin

5.5 8.59 Levofloxacin

5.45 6.2  Moxifloxacin

5.69 9.42 Nalidixic acid

5.95 4.68 Norfloxacin

5.77 8.68 Pefloxacin

5.66 6.47

It is noted that antibiotic uptake is a complex function of permeabilityand efflux. The impact of bicarbonate on the pH gradient likely alsoimpacts drug efflux, particularly in Gram-negative bacteria. Manymultidrug efflux pumps depend on the PMF, where energy from the protongradient is harnessed to expel drugs from the cell, such as theResistance-Nodulation-Division (RND)-system AcrAB-TolC in E. coli. Itwas assessed whether consistent with a role in dissipating ΔpH,bicarbonate would reduce efflux activity. The potentiation ofdirithromycin, for example, by bicarbonate was lost in a strain lackingthe outer membrane channel of this tripartite efflux system (ΔtolC)(FIG. 11 , Panel c), indicating it was inhibition of efflux by sodiumbicarbonate that led to its enhanced activity. Although macrolideantibiotics are thought to be of little value for the treatment ofGram-negative bacteria due to their diminished accumulation thesestudies show that in the bicarbonate-rich environment of the host,energy-depended efflux systems may be less effective than predicted byconventional in vitro MIC determinations. Panel c shows that lack of theouter membrane tripartite pump, tolC, causes a loss of potentiation ofsodium bicarbonate towards dirithromycin in E. coli. Shown are potencyanalyses of dirithromycin against: wild-type E. coli in MHB (1);wild-type E. coli in MHB supplemented with 25 mM sodium bicarbonate (2);ΔtolC in MHB (3); ΔtolC in MHB supplemented with 25 mM sodiumbicarbonate (4). Averages of triplicate experiments are shown.

Inhibition of cell wall synthesis was attenuated in the presence ofsodium bicarbonate in E. coli only 2-4 fold on average, but thissuppression was more pronounced in S. aureus, which is generally moresusceptible to cell wall synthesis inhibitors than Gram-negativebacteria. Where cell wall-active compounds are most effective onactively dividing bacteria, suppression of the action of the cidalantibiotics, β-lactams and cephalosporins, for example, may be due toreduced respiratory energy production to fuel growth in the presence ofPMF-altering concentrations of bicarbonate (Lobritz, M. A. et al.Antibiotic efficacy is linked to bacterial cellular respiration. ProcNatl Acad Sci USA 112, 8173-8180, doi:10.1073/pnas.1509743112 (2015)).Accordingly, a significant effect on cellular respiration (70%reduction) in E. coli was observed, when treated with 25 mM sodiumbicarbonate (FIG. 14 ). Specifically shown in FIG. 14 is the effect of25 mM sodium bicarbonate on the reduction of2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT)to INT-formazan. Open bars indicate the formation of formazan at t=0 asread at 490 nm. Grey bars represent the formation of formazan following60 min incubation.

Consistent with this finding, E. coli grown in high concentrations ofsodium bicarbonate exhibited a delayed lag phase, indicating loweredmetabolic resources (FIG. 15 ). FIG. 15 shows the growth curve of E.coli grown in the presence of varying concentrations of sodiumbicarbonate. Growth curve measurements were performed in a microtiterplate and optical density read every 10 mins in a Tecan infinite M1000Pro with shaking intervals before readings.

Further, intracellular ATP levels, which are produced via theF₀F₁-ATPase utilizing PMF, were reduced by ˜30% in sodiumbicarbonate-treated E. coli compared to the untreated control (FIG. 16). FIG. 16 shows the effect of 25 mM sodium bicarbonate on intracellularATP levels, measured by a luciferin-luciferase bioluminescence assay.Shown is the relative fluorescence units for untreated S. aureus cells(white bar) and for 25 mM bicarbonate treated S. aureus cells (grey).

Overall, these experiments indicate that bicarbonate is a bacteriostaticcompound that perturbs cellular respiration and reduces the activity ofbactericidal antibiotics that require actively growing bacteria foractivity.

Example 15: Effects of Gene Deletions in E. coli on the Ability ofBicarbonate to Reduce Growth

The mode of action of bicarbonate on E. coli physiology was furtherinvestigated. The impact of 25 mM sodium bicarbonate on an ordered E.coli gene-deletion collection of ˜4,000 strains was assessed. Sodiumbicarbonate reduced the growth of 28 deletion strains. The missing genesencoded proteins involved in redox reactions and oxidative stressresponses (FIG. 17 , Panel a, Table 9). Among them was dsbB, whose geneproduct is required to maintain disulfide bonds in periplasmic enzymesat extreme pHs, and the gene encoding the sigma factor RpoS thatregulates several components of resistance to both acid and base.Deletion in the gene cydX, coding for a cytochrome oxidase, causedsensitization to bicarbonate. Deletion in the gene nhaA, which encodes aNa⁺:H⁺ antiporter that has a major role in sodium ion and alkaline pHhomeostasis in E. coli and many enterobacteria, sensitized cells tobicarbonate. It was observed that a defect in proton expulsion enhancedthe growth inhibition by bicarbonate. Deletion of cya was alsosensitized to bicarbonate. Overall, it was observed that gene deletionssensitized to bicarbonate involved pH-related processes, through protonexpulsion or stress responses, that when deleted amplify bicarbonate'saction on the pH gradient across the inner membrane.

FIG. 17 , Panel a: Keio collection was exposed to 25 mM bicarbonate for15 hours at 37° C., and sick/lethal interactions were assessed using amultiplicative approach (French, S. et al. A robust platform forchemical genomics in bacterial systems. Mol Biol Cell 27, 1015-1025,doi:10.1091/mbc.E15-08-0573 (2016)). Shown alongside an index plot ofthe chemical-genetic interactions are the 15 mutations that moststrongly enhanced the activity of bicarbonate (displayed as1-interaction score).

TABLE 9 Genetic enhancers (Keio collection) of growth inhibition by 25mM bicarbonate. Strains were exposed to bicarbonate for 15 hours incation-adjusted MHB broth, and a multiplicative approach was used todetermine the sick or lethal effects on each strain. Shown here are theoutliers from FIG. 17, Panel a, alongside their gene products, asannotated from EcoCyc⁵. Deletion strain Gene description appX smallouter membrane protein cyaA adenylate cyclase cydX cytochrome bd Iterminal oxidase - CydX subunit degP serine protease Do dnaT primosomalprotein DnaT dsbB protein disulfide oxidoreductase envC EnvC divisomeassociated factor, activator of peptidoglycan hydrolases fur Furtranscriptional dual regulator galE UDP-glucose 4-epimerase glnAadenylyl-[glutamine synthetase], glutamine synthetase hfq RNA-bindingprotein that affects many cellular processes; homolog of mammalianSm/Sm-like proteins lpoB outer membrane lipoprotein - activator of MrcBactivity mgrB negative feedback regulator of the PhoQP system nhaANa⁺:H⁺ antiporter NhaA pgi phosphoglucose isomerase recB RecB rodZtransmembrane component of cytoskeleton rplA 50S ribosomal subunitprotein L1 rpmF 50S ribosomal subunit protein L32 rpoS RNA polymerase,sigma S (sigma 38) factor rpsT 30S ribosomal subunit protein S20 rsgAribosome small subunit-dependent GTPase A sapA periplasmic bindingprotein SapA of predicted ABC trans- porter treA periplasmic trehalaseubiF 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone hydro- xylase ubiH2-octaprenyl-6-methoxyphenol hydroxylase ybbY putative transportprotein, nucleobase:cation symporter-2 (NCS2) family ybcO DLP12prophage; predicted protein yciB inner membrane protein

Example 16: Adaptive Strategies by E. coli in Response to SodiumBicarbonate

The action of sodium bicarbonate on E. coli was further assessed byanalyzing promoter activity in response to 25 mM sodium bicarbonateusing a genome-scale, promoter-reporter library where nearly all of thepromoters in E. coli have been transcriptionally fused to gfp (FIG. 17 ,Panel b, Table 10).

Shown in FIG. 17 , Panel b are the responses of a genome-scale GFPpromoter library (Zaslaver, A. et al. A comprehensive library offluorescent transcriptional reporters for Escherichia coli. Nat Methods3, 623-628, doi:10.1038/nmeth895 (2006)), to 25 mM bicarbonate.Highlighted are promoters with increased and decreased expression. Ofnote are the genes nhaA and hfq, that were strong enhancers of growthinhibition by bicarbonate and were differentially expressed inbicarbonate containing medium.

TABLE 10 List of promoters from the GFP promoter-fusion library thatdemonstrated increased or decreased promoter activity in the presence of25 mM bicarbonate. Activity was assessed using the pipeline of Zaslaveret al². Shown here are the promoters from FIG. 17, Panel b, alongsidetheir gene products, as annotated from EcoCyc⁵. Name Product Increasedpromoter activity ais Predicted lipopolysaccharide coreheptose(II)-phosphate phosphatase alsB D-allose ABC transporter -periplasmic binding protein asd Aspartate semialdehyde dehydrogenasecspI Qin prophage; cold shock protein dinG ATP-dependent helicase dmlAD-malate/3-isopropylmalate dehydrogenase (decarboxylating) dusAtRNA-dihydrouridine synthase A dusB tRNA-dihydrouridine synthase B entDPhosphopantetheinyl transferase erpA Essential respiratory protein AfetA ABC transporter with a role in iron homeostasis - ATP- bindingsubunit glyU tRNA-glyU htrL Involved in lipopolysaccharide biosynthesisiscR IscR DNA-binding transcriptional dual regulator kefF Regulator ofKefC-mediated potassium transport and quinone oxidoreductase lpxCUDP-3-O-acyl-N-acetylglucosamine deacetylase mltC Membrane-bound lyticmurein transglycosylase C murJ Lipid II flippase mutY Adenineglycosylase; G.C --> T.A transversions nhaA Na+:H+ antiporter NhaA potFPutrescine ABC transporter - periplasmic binding protein rcsC RcsCsensory histidine kinase - asp875 phosphorylated rfaH RfaHtranscriptional antiterminator rplN 50S ribosomal subunit protein L14rplY 50S ribosomal subunit protein L25 rpsJ 30S ribosomal subunitprotein S10 rpsM 30S ribosomal subunit protein S13 rpsO 30S ribosomalsubunit protein S15 rpsP 30S ribosomal subunit protein S16 rsfSRibosomal silencing factor smpB Small protein B sppA Protease IV, asignal peptide peptidase tig Chaperone protein Tig; trigger factor trxAOxidized thioredoxin, thioredoxin 1 ttcA tRNA C32 thiolase waaZ Proteininvolved in KdoIII attachment during lipopolysaccha- ride corebiosynthesis yacG DNA gyrase inhibitor YacG ybaA Conserved protein ybaBConserved DNA-binding protein ybfE LexA-regulated protein ycgM Predictedisomerase/hydrolase ydeA Arabinose exporter yeiE LYSR-typetranscriptional regulator yejL Conserved protein yidH Conserved innermembrane protein yjdI Conserved protein yncE Conserved protein yphGConserved protein zraP Zinc responsive, periplasmic protein withchaperone activity Decreased promoter activity aspU tRNA-aspU bioBBiotin synthase deaD DeaD, DEAD-box RNA helicase fadE Acyl-CoAdehydrogenase folD Bifunctional 5,10-methylene-tetrahydrofolate dehydro-genase/5,10-methylene-tetrahydrofolate cyclohydrolase fprFlavodoxin-NADP+ reductase/ferredoxin-NADP+ reductase ftp Flavintransferase ftsZ Essential cell division protein FtsZ gcvA GcvADNA-binding transcriptional dual regulator glnA Adenylyl-[glutaminesynthetase], glutamine synthetase glrK GlrK sensory histidine kinase -phosphorylated, GlrK sensory histidine kinase hemA Glutamyl-tRNAreductase hfq RNA-binding protein that affects many cellular processes;homolog of mammalian Sm/Sm-like proteins hofM Protein involved inutilization of DNA as a carbon source hscC Hsc62, Hsp70 familychaperone, binds to RpoD and inhibits transcription lysO L-lysineexporter metZ TRNA-fMet1 pepQ Xaa-Pro dipeptidase polB DNA polymerase IIprlF PrlF antitoxin radD Predicted ATP-dependent helicase; implicated inDNA repair rapA RNA polymerase-binding ATPase and RNAP recycling factorrhaD Rhamnulose-1-phosphate aldolase rpmI 50S ribosomal subunit proteinL35 rrfG rrfG 5S ribosomal RNA rrlH rrlH 23S ribosomal RNA sbcBExonuclease I, 3′ --> 5′ specific; deoxyribophosphodiesterase tcdA tRNAthreonylcarbamoyladenosine dehydratase yahK Aldehyde reductase,NADPH-dependent ybiO Mechanosensitive channel YbiO ydbC Predictedoxidoreductase, NAD(P)-binding yegW Predicted DNA-bindingtranscriptional regulator ygbI Predicted DNA-binding transcriptionalregulator, DEOR-type yiaT Outer membrane protein YiaT yieH6-Phosphogluconate phosphatase yjbF Predicted lipoprotein yjfY Putativeprotein ykgF Predicted amino acid dehydrogenase with NAD(P)-bindingdomain and ferridoxin-like domain ykgJ Predicted ferredoxin ynfC YnfClipoprotein yodB Predicted cytochrome ypdA YbdA sensory histidinekinase - his371 phosphorylated

Changes in promoter activity that reflected adaptive strategies by thebacterium to maintain pH homeostasis were observed. Promoter activityfor a large number of substrate/proton antiporters was repressed in thepresence of bicarbonate. Promoter activity for nhaA was enhanced in thepresence of bicarbonate.

Upon treatment with bicarbonate, cytoplasmic pH, which began at ˜7.5,endured a rapid cytoplasmic alkalinization, as measured by BCECF-AM(FIG. 20 ). FIG. 20 shows changes in intracellular pH upon treatmentwith bicarbonate. S. aureus cells were loaded with the pH sensitive dyeBCECF-AM and were washed and resuspended in PBS. Following baselinereadings, PBS (grey circles) or 25 mM sodium bicarbonate (black circles)were added at the arrow and fluorescence measured over time. A standardcurve for intracellular pH calibration was used to calculateintracellular pH.

The expression of a number of inner membrane protein/transporters wasdifferentially regulated in the presence of bicarbonate. Decreasedpromoter activity was also observed for many ATP-dependent processes,likely as an adaptive effort to conserve energy.

These data indicate that overall, E. coli adaptation to bicarbonateinvolved strategies to respond to periplasmic pH changes, increasemembrane potential and preserve energy.

Example 17: The Effect of Bicarbonate on the Activity of Molecules thatSelectively Perturb Δψ

It was assessed whether bicarbonate enhances the activity of moleculesthat selectively perturb Δψ. To test this, the following Δψ dissipaterswere combined with sodium bicarbonate: valinomycin, a selectivepotassium ionophore, as well as compounds that have previouslycharacterized as dissipaters of Δψ, namely I1, I2 and I3 (Farha, M. A.,Verschoor, C. P., Bowdish, D. & Brown, E. D. Collapsing the protonmotive force to identify synergistic combinations against Staphylococcusaureus. Chemistry & biology 20, 1168-1178,doi:10.1016/j.chembiol.2013.07.006 (2013)) and loperamide (Ejim, L. etal. Combinations of antibiotics and nonantibiotic drugs enhanceantimicrobial efficacy. Nature chemical biology 7, 348-350,doi:10.1038/nchembio.559 (2011).

All combinations yielded synergistic interactions, consistent with therole of bicarbonate as a selective dissipater of ΔpH (FIG. 18 ). FIG. 18, Panels a-c show a microdilution checkerboard analyses for sodiumbicarbonate and molecules shown to dissipate ΔΨ; Panel a) valinomycin inS. aureus; Panel b) loperamide in E. coli; and Panel c) molecules I1-3in S. aureus. All checkerboards display synergistic interactions.

This indicates that membrane active agents that target Δψ, whilefrequently eschewed in drug discovery efforts for potentialcytotoxicity, can have superior activity in the bicarbonate-richenvironment of the host.

Example 18: The Effect of Bicarbonate is not an Effect of Changes in pH

PMF is driven in part by a transmembrane gradient where the periplasmicside of the membrane has a greater concentration of protons. Thus, theaddition of buffering agents to alter the external pH can have asignificant effect on PMF. To assess the impact of such a perturbation,trisodium phosphate (Na₃PO₄) was added, which increased the pH of themedia by 3 units and also potentiated the activity of dirithromycin.Adjusting the pH back to neutrality, however, led to a loss of synergy(FIG. 19 ). FIG. 19 , Panels a-b show the effect of pH-adjusting mediaon the combination of dirithromycin with trisodium phosphate. Shown aremicrodilution checkerboard analyses for dirithromycin and trisodiumphosphate when Panel a) pH is not adjusted to 7.2 (pH ˜10) and whenPanel b) pH of the medium is adjusted to 7.2. Conversely,supplementation with sodium bicarbonate has little impact on the pH.Steps were taken throughout the studies to confirm that bicarbonatesupplemented media were at pH 7.4, and adjusted where necessary. Thebicarbonate effect is not a trivial consequence of the pH of the media.Supplementation of the media with a variety of buffer systems indicatedthat potentiation was unique to bicarbonate.

Consistent with the idea that bicarbonate is acting extra-cytoplasmic,presumably in the periplasmic space, no difference in bicarbonate'sability to potentiate dirithromycin in a bicarbonatetransporter-deficient strain (ΔychM) and in a wild-type strain wasobserved (FIG. 21 ). FIG. 21 , Panels a-b show the growth inhibition bydirithromycin and sodium bicarbonate. Bacterial strains were wild typeE. coli in Panel a; and ΔychM in Panel b.

Example 19: Data

With respect to pentamidine, taken together these examples show thatwhile pentamidine's in vitro MIC's in standard microbiological media,enforced by the Clinical & Laboratory Standards Institute, discouragesits clinical utility, an unforeseen dependence on media conditionsrevealed an enhanced in vitro activity. The enhanced antibacterialactivity of pentamidine depended on the presence of an ionic milieu thatis comparable to the conditions found in mammalian tissues andspecifically on the presence of bicarbonate. Indeed, the antibacterialactivity of pentamidine was potentiated with increasing concentrationsof sodium bicarbonate. Pentamidine activity was found to be antagonizedin the presence of NaCl, which is present in high concentrations instandard microbiological media. In the presence of a counter ion such asbicarbonate, however, the antibacterial activity of pentamidine waspotentiated on average 40-fold against Gram-negative organisms and50-fold against Gram-positive organisms. Bicarbonate is ubiquitous inthe mammalian body and present in high concentrations in varioustissues.

Consistently, an unforeseeable efficacy for pentamidine in clearing anA. baumannii systemic infection in mice was observed for the first time.The consequence of secondary factors affecting pentamidine'santibacterial activity is consistent with the significant in vivoactivity observed.

In addition to diamidines, like pentamidine, many conventionalantibiotics also displayed an interaction with bicarbonate. Remarkably,varying classes of conventional antibiotics displayed significantpotentiation (macrolides, and some fluoroquinolones) in the presence ofphysiological concentrations of bicarbonate (25 mM). Significantly, notall conventional antibiotics displayed a synergistic interaction withbicarbonate as some antibiotics resulted in significant suppression ofactivity (e.g. tetracyclines, some fluoroquinolones, cell wall activeantibiotics, amino-coumarins) in the presence of physiologicalconcentrations of bicarbonate (25 mM).

The investigations into the mode of action of bicarbonate revealed anability to dissipate the pH gradient of the proton motive force acrossthe cytoplasmic membrane. In doing so, bicarbonate suppresses the entryof antibiotics that are driven by the pH gradient and enhances the entryof antibiotics that are driven by the opposing and compensatorycomponent, the membrane potential. Further, by disrupting the energeticsacross the membrane, bicarbonate also disrupts energy-dependent effluxsystems thus further enhancing the accumulation of antibiotics that areactively effluxed. In the case of pentamidine, the latter disrupts themembrane potential across the membrane, thus when used with bicarbonate,both components that make up the proton motive force are synergisticallytargeted. This phenomenon was also observed with other various smallmolecules that dissipate membrane potential when used with bicarbonate.

Like bacterial cells, yeast and fungal cells have a cytoplasm and amembrane surrounded by a cell wall. Within the plasma membrane is achemiosmotic mechanism that is very similar to that of bacteria. Infact, these energetics mechanisms underlie the function of nearly allliving organisms. Indeed, energetics via components of proton motiveforce in yeast and fungi is very similar to that of bacteria and runs atsimilar values (−150 to −200 mV).

Bicarbonate is present in all body fluids and organs and plays acritical role in maintaining acid-base balance in the human body. Here,to better understand the bacteriostatic mechanism of bicarbonate, itsinteraction with antimicrobial agents (e.g. antibiotics) of varyingmechanisms of action was studied. Many classes of conventionalantibiotics displayed significant potentiation or suppression in thepresence of physiological concentrations of bicarbonate. Allinteractions pointed to a mechanism whereby the bicarbonate ion causesperturbation of the pH gradient of proton motive force (PMF) across thecytoplasmic membrane. The product of cellular respiration, PMF,describes the electrochemical potential at the cytoplasmic membrane thatis composed of an electrical potential (Δψ, negative inside) and aproton gradient (ΔpH, acidic outside). This electrochemical potentialcrucially underpins energy production so that bacterial cells work tomaintain a constant PMF (Bakker, E. P. & Mangerich, W. E.. J Bacteriol1981, 147: 820-826). Agents that perturb either Δψ or ΔpH are growthinhibitory and prompt a compensatory increase in the other component inorder to maintain PMF. Further, synergy in growth inhibition is observedwhen an agent active on the electrical potential is combined with anagent that targets the proton gradient (Farha et al, Chemistry andBiology 2013, 20:1168-78). Thus in dissipating the pH gradient,bicarbonate had enhancing effects on other antibacterial compoundsthrough distinct mechanisms: (a) Bicarbonate dissipated the pH gradientacross the cytoplasmic membrane and led to an increase in thecompensatory component, the membrane potential. For antibiotics whoseentries are dependent on the membrane potential, an enhancement ofgrowth inhibition was observed in the presence of sodium bicarbonate,consistent with an increase in antibiotic entry. Further, by disruptingthe energetics across the membrane, bicarbonate also disruptsenergy-dependent efflux thus potentiating the activity of effluxsubstrates (e.g. macrolides). In these instances, it was observed thatbicarbonate led to an increase in intracellular concentration of theantibiotic. (b) In an alternate mechanism, bicarbonate enhanced thosecompounds that disrupt membrane potential component as a primarymechanism of action. These activities were potentiated via a synergisticcollapse of both components of PMF, Δψ by the antibiotic and ΔpH bybicarbonate. In all, by altering the cell's transmembrane pH gradient,bicarbonate potentiates antibiotic activity by increasing the effectiveintracellular levels of various antibiotics or enhancing their abilityto collapse PMF, including pentamidine, an overlooked antibacterialthought to lack in vivo antibacterial activity. Furthermore, it wasobserved that bicarbonate works together with components of innateimmunity (innate immunity factors) to inhibit the growth of bacterialpathogens. In sum, these data implicate bicarbonate as an overlookedpotentiator of host immunity in the defense against pathogens. Thisstudy suggests that bicarbonate is an overlooked immune factor that maylead to unrecognized in vivo activities of clinically useful antibioticsand a potential natural and innocuous additive in the design of noveltherapeutic strategies.

Example 20: The Effect of Bicarbonate on the Activity of Antibiotics

The effect of sodium bicarbonate on the activity of variousantibacterial agents was investigated. Clinical isolates of variousbacteria were obtained from the American Type Culture Collection (ATCC)and the International Health Management Associates (IHMA). Fractionalinhibitory concentration indices (FICIs) were determined by setting upstandard checkerboard broth microdilution assays in 96-well microtiterplates with serially diluted 8 (or 10) concentrations of each drug(sodium bicarbonate and the antibacterial agent). The protocol forcheckerboard analyses was based on the Clinical & Laboratory StandardsInstitute (CLSI) guidelines. Plates were incubated at 37° C. for 18hours, and optical density was read at 600 nm. At least 3 replicateswere done for each query compound. Graphical results of these assays areshown in FIGS. 22-25 .

The minimum concentration for inhibition (MIC) for each drug was thelowest drug concentration showing <10% growth. The FIC for each drug wascalculated as the concentration of drug in the presence of the co-drugfor a well showing <10% growth, divided by the MIC for that drug, asshown in the equation below. The FIC index (FICI) is the sum of the twoFICs. Chemical-chemical interactions with ΣFIC of less or equal to 0.5were deemed synergistic.

Fractional Inhibitory Concentration (FIC)=[X]/MIC_(X), where [X] is thelowest inhibitory concentration of drug in the presence of the co-drug.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Where a term provided herein is found to be defineddifferently in a document incorporated herein by reference, thedefinition provided herein is to serve as the definition for the term.

The invention claimed is:
 1. A method for inhibiting the growth of abacterium, comprising contacting the bacterium with an effective amountof (i) bicarbonate and (ii) an antimicrobial agent; wherein theantimicrobial agent is azithromycin, and the effective amount of thebicarbonate is a concentration of about 1 mM to about 150 mM.
 2. Amethod of treating or preventing a microbial infection, comprisingadministering to a subject in need thereof an effective amount of (i)bicarbonate and (ii) an antimicrobial agent; wherein the microbialinfection is an infection by a bacterium; wherein the antimicrobialagent is azithromycin; and wherein the effective amount of bicarbonateis a concentration of about 1 mM to about 150 mM.
 3. The method of claim1 or 2, wherein the bicarbonate is a component of a buffer.
 4. Themethod of claim 1 or 2, wherein the bicarbonate is sodium bicarbonate,ammonium bicarbonate, lithium bicarbonate, potassium bicarbonate,magnesium bicarbonate, calcium bicarbonate or zinc bicarbonate.
 5. Themethod of claim 1 or 2, wherein the amount of the bicarbonate is aphysiological concentration of the bicarbonate.
 6. The method of claim 1or 2, wherein the bicarbonate is present in a composition being about0.01 wt % to about 1 wt % of bicarbonate.
 7. The method of claim 1 or 2,wherein the bicarbonate and the antimicrobial agent are present in acomposition.
 8. The method of claim 1 or 2, wherein the bicarbonate ispresent in a first composition and the antimicrobial agent is present ina second composition.
 9. The method of claim 1 or 2, wherein thebacterium is: (a) a rod-shaped bacterium, a sphere-shaped bacterium, aspiral-shaped bacterium, a filamentous bacterium, a pleomorphicbacterium or a rectangular bacterium; (b) a facultative anaerobe, anobligate aerobe or an obligate anaerobe; (c) a Gram positive bacteriumor a Gram negative bacterium; (d) a Gram positive rod-shaped bacterium;(e) a Gram negative sphere-shaped bacterium; or (f) a species ofAcinetobacter, Actinomyces, Aerococcus, Agrobacterium, Anaplasma,Azorhizobium, Azotobacter, Bacillus, Bacteroides, Bartonella,Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium,Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium,Coxiella, Ehrlichia, Enterobacter, Enterococcus, Escherichia,Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobatter,Klebsiella, Lactobacillus, Lactococcus, Legionella, Listeria,Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium,Mycoplasma, Neisseria, Pasteurella, Pediococcus, Peptostreptococcus,Porphyromonas, Prevotella, Pseudomonas, Rhizobium, Rickettsia,Rochalimaea, Rothia, Salmonella, Serratia, Shigella, Sarcina, Spirillum,Spirochaetes, Staphylococcus, Stenotrophomonas, Streptobacillus,Streptococcus, Tetragenococcus, Treponema, Vibrio, Viridans, Wolbachiaor Yersinia.
 10. The method of claim 1 or 2, wherein the bacterium isAcetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii,Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasmaphagocytophilum, Azorhizobium caulinodans, Azotobacter vinelandii,Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillusfusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillusmycoides, Bacillus stearothermophilus, Bacillus subtilis, BacillusThuringiensis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroidesmelaninogenicus, Bartonella henselae, Bartonella Quintana, Bordetellabronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucellaabortus, Brucella melitensis, Brucella suis, Burkholderia mallei,Burkholderia pseudomallei, Burkholderia cepacia, Calymmatobacteriumgranulomatis, Campylobacter coli, Campylobacter fetus, Campylobacterjejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophilapneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridiumdifficile, Clostridium perfringens, Clostridium tetani, Corynebacteriumdiphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Ehrlichiachafeensis, Enterobacter cloacae, Enterococcus avium, Enterococcusdurans, Enterococcus faecalis, Enterococcus faecium, Enterococcusgalllinarum, Enterococcus maloratus, Escherichia coli, Francisellatularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilusducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophiluspertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiellapneumoniae, Lactobacillus acidophilus, Lactobacillus bulgaricus,Lactobacillus casei, Lactococcus lactis, Legionella pneumophila,Listeria monocytogenes, Methanobacterium extroquens, Microbacteriummultiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacteriumavium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacteriumintracellulare, Mycobacterium leprae, Mycobacterium lepraemurium,Mycobacterium phlei, Mycobacterium smegmatis, Mycobacteriumtuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasmahominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseriagonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurellatularensis, Peptostreptococcus, Porphyromonas gingivalis, Prevotellamelaninogenica, Pseudomonas aeruginosa, Rhizobium radiobacter,Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana,Rickettsia rickettsii, Rickettsia trachomas, Rochalimaea henselae,Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis,Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigelladysenteriae, Spirillum volutans, Staphylococcus aureus, Staphylococcusepidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae,Streptococcus avium, Streptococcus bovis, Streptococcus cricetus,Streptococcus faceium, Streptococcus faecalis, Streptococcus fetus,Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior,Streptococcus mitis, Streptococcus mutans, Streptococcus oxalis,Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus,Streptococcus sahvarius, Streptococcus sanguis, Streptococcus sobrinus,Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma,Vibrio parahaemolyticus, Vibrio vulnificus, Viridans streptococci,Wolbachia, Yersinia enterocolitica, Yersinia pestis or Yersiniapseudotuberculosis.
 11. The method of claim 10, wherein the bacterium ismethicillin-resistant Staphylococcus aureus (MRSA) ormethicillin-sensitive Staphylococcus aureus (MSSA).
 12. The method ofclaim 1, wherein the bacterium is present on a substrate.
 13. The methodof claim 12, wherein the substrate is glass, metal, plastic, latex,ceramic, cement, wood, grout or stone.
 14. The method of claim 2,wherein the infection is a pulmonary, lung, otic, oral, nasal, sinus,ophthalmic, intraocular, dermal, cardiovascular, kidney, urinary,gastro-intestinal, rectal, vaginal or neurological infection.
 15. Themethod of claim 1 or 2, wherein the bicarbonate or the antimicrobialagent is present in a solution, a gel, a cream, a lotion, a liquidsuspension, an aerosol, a nebulized spray, an ointment, drops or apatch.
 16. The method of claim 2, wherein the bicarbonate or theantimicrobial agent is administered as an ophthalmic topical solution orgel, or by topical, dermal, transdermal, subconjunctival, periocular,retrobulbar, subtenon, intracameral, intravitreal, intraocular,subretinal, juxtascleral or supra-choroidal administration.
 17. Themethod of claim 7, wherein the bicarbonate is present in the compositionat a concentration of about 20 mM to about 100 mM.
 18. The method ofclaim 7, wherein the composition further comprises a pharmaceuticallyacceptable carrier, diluent or excipient.
 19. The method of claim 17,wherein the bicarbonate is a component of a buffer.
 20. The method ofclaim 17, wherein the bicarbonate is sodium bicarbonate, ammoniumbicarbonate, lithium bicarbonate, potassium bicarbonate, magnesiumbicarbonate, calcium bicarbonate or zinc bicarbonate.
 21. The method ofclaim 17, wherein the bicarbonate is present in the composition at aconcentration of about 25 mM to about 50 mM.
 22. The method of claim 17,wherein: the bicarbonate is present in the composition at aconcentration of about 35 mM to about 50 mM; and the azithromycin ispresent in the composition at a concentration of about 1 μg/mL or about2 μg/mL.
 23. The method of claim 18, wherein the pharmaceuticallyacceptable carrier, diluent or excipient is suitable for ocularadministration.
 24. The method of claim 7, wherein the compositionfurther comprises a thickener.
 25. The method of claim 24, wherein thethickener is methyl cellulose, lactose, mannitol, maltose, hyaluronicacid, sodium hyaluronate, potassium hyaluronate, chondroitin sulfate,sodium polyacrylate, carboxyvinyl polymer, crosslinked polyacrylate,polyvinyl alcohol, polyvinyl pyrrolidone, hydroxy propylmethylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose orhydroxy propyl cellulose.
 26. The method of claim 7, wherein thecomposition has a pH of 7.4.
 27. The method of claim 7, wherein thebicarbonate decreases the growth of a bacterium in response to theantimicrobial agent; and wherein the bacterium is Pseudomonasaeruginosa, Staphylococcus aureus or Streptococcus pneumoniae.
 28. Themethod of claim 27, wherein the Staphylococcus aureus ismethicillin-resistant Staphylococcus aureus (MRSA).
 29. The method ofclaim 27, wherein the bicarbonate decreases the growth of the bacteriumin response to the antimicrobial agent by 2-fold to 30-fold.